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  • 101
    ISSN: 0730-2312
    Keywords: osteoprogenitors ; marrow-stroma ; alkaline phosphatase ; bisphosphonates ; cell proliferation ; mineralization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bisphosphonates (BPs) are inhibitors of bone resorption and soft tissue calcification. The biological effects of the BPs in calcium-related disorders are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and/or osteoblasts through a variety of biochemical pathways. Structural differences account for the considerable differences in the pharmacological activity of BPs. We compared the effects of two structurally different compounds, alendronate and 2-(3′-dimethylaminopyrazinio)ethylidene-1,1-bisphosphonic acid betaine (VS-6), in an osteoprogenitor differentiation system. The BPs were examined in a bone marrow stromal-cell culture system, which normally results in osteoprogenitor differentiation. The drugs were present in the cultures from days 2 to 11 of osteogenic stimulation, a period estimated as being comparable to the end of proliferation and the matrix-maturation stages. We found that the two different BPs have opposing effects on specific alkaline phosphatase (ALP) activity, on stromal-cell proliferation, and on cell-mediated mineralization. These BPs differentially interact with cell-associated phosphohydrolysis, particularly at a concentration of 10-2 of ALP Km, in which alendronate inhibits whereas VS-6 did not inhibit phosphatase activity. VS-6 treatment resulted in similar and significantly increased mineralization at 10 and 1 μM drug concentrations, respectively. In contrast, mineralization was similar to control, and significantly decreased at 10 and 1 μM drug concentrations, respectively, under alendronate treatment. J. Cell. Biochem. 68:186-194, 1998. © 1998 Wiley-Liss, Inc.
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  • 102
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 200-212 
    ISSN: 0730-2312
    Keywords: polyamines ; chromatin structure ; micrococcal nuclease ; cell cycle ; apoptosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc- cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G1-early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G2 phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression. J. Cell Biochem. 68:200-212, 1998. © 1998 Wiley-Liss, Inc.
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  • 103
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 213-225 
    ISSN: 0730-2312
    Keywords: glutamine ; glutamate ; mitochondria ; metabolism ; HeLa cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The oxidative metabolism of glutamine in HeLa cells was investigated using intact cells and isolated mitochondria. The concentrations of the cytoplasmic amino acids were found to be aspartate, 8.0 mM; glutamate, 22.2 mM; glutamine, 11.3 mM; glycine, 9.8 mM; taurine, 2.3 mM; and alanine, 〈1 mM. Incubation of the cells with [14C]glutamine gave steady-state recoveries of 14C-label (estimated as exogenous glutamine) in the glutamine, glutamate, and aspartate pools, of 103%, 80%, and 25%, respectively, indicating that glutamine synthetase activity was absent and that a significant proportion of glutamate oxidation proceeded through aspartate aminotransferase. No label was detected in the alanine pool, suggesting that alanine aminotransferase activity was low in these cells. The clearance rate of [14C]glutamine through the cellular compartment was 65 nmol/min per mg protein. There was a 28 s delay after [14C]glutamine was added to the cell before 14C-label was incorporated into the cytoplasm, while the formation of glutamate commenced 10 s later.Aspartate was the major metabolite formed when the mitochondria were incubated in a medium containing either glutamine, glutamate, or glutamate plus malate. The transaminase inhibitor AOA inhibited both aspartate efflux from the mitochondria and respiration. The addition of 2-oxoglutarate failed to relieve glutamate plus malate respiration, indicating that 2-oxoglutarate is part of a well-coupled truncated cycle, of which aspartate aminotransferase has been shown to be a component [Parlo and Coleman (1984): J Biol Chem 259:9997-10003]. This was confirmed by the observation that, although it inhibited respiration, AOA did not affect the efflux of citrate from the mitochondria. Thus citrate does not appear to be a cycle component and is directly transported to the medium. Therefore, it was concluded that the truncated TCA cycle in HeLa cells is the result of both a low rate of citrate synthesis and an active citrate transporter. DNP (10 μM) induced a state III-like respiration only in the presence of succinate, which supports the evidence that NAD-linked dehydrogenases were not coupled to respiration, and suggests that these mitochondria may have a defect in complex I of the electron transport chain. Arising from the present results with HeLa cells and results extant in the literature, it has been proposed that a major regulating mechanism for the flux of glutamate carbon in tumour cells is the competitive inhibition exerted by 2-oxoglutarate on aspartate and alanine aminotransferases. This has been discussed and applied to the data. J. Cell. Biochem. 68:213-225, 1998. © 1998 Wiley-Liss, Inc.
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  • 104
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 247-258 
    ISSN: 0730-2312
    Keywords: SMCs ; bFGF ; collagen fibril structure ; mRNA ; atherosclerotic lesion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular smooth muscle cells (SMCs), the major cellular constituent of an artery, synthesize the bulk of fibrillar collagens, including type V/XI, which regulates heterotypic collagen fibril assembly. Basic fibroblast growth factor (bFGF) is a heparin-binding polypeptide growth factor that has been implicated in important events during the development of atherosclerosis, such as early intimal SMC proliferation. Here we have investigated the effects of bFGF on aortic SMC expression of type V/XI collagen. Treatment of exponentially growing or serum-deprived subconfluent cultures of bovine aortic SMCs with bFGF decreased the steady-state levels of the mRNAs for collagen type V/XI, including α1(V), α2(V), and α1(XI). The effect of bFGF was time dependent with a two- and a fourfold decrease in α2(V) mRNA observed after treatment for 24 and 48 h, respectively. This decrease resulted from a drop in the rate of α2(V) gene transcription; no change was observed in the stability of the α2(V) mRNA. Furthermore, accumulation of collagen protein decreased upon bFGF treatment. As expected, treatment with bFGF increased the rate of proliferation of serum-deprived SMCs, as judged by DNA content in the cultures, thymidine incorporation, and steady-state mRNA levels of the S-phase-expressed histone H3.2. These results suggest that bFGF plays an important role in the regulation of collagen fibril structure, with potential implications for the development and organization of an atherosclerotic lesion. J. Cell. Biochem. 68:247-258, 1998. © 1998 Wiley-Liss, Inc.
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  • 105
    ISSN: 0730-2312
    Keywords: transforming growth factor-β ; tumor necrosis factor-α ; phospholipase A2 ; arachidonic acid ; AACOCF3 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The steroid derivative 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a regulator of bone biology, and there is evidence that 1,25(OH)2D3 modulates arachidonic acid metabolism in osteoblastic cell model systems and in bone organ cultures. In the present studies, 1,25(OH)2D3 decreased prostaglandin (PG) biosynthesis by normal adult human osteoblast-like (hOB) cell cultures by about 30%. The decrease was observed under basal incubation conditions, or in specimens stimulated by transforming growth factor-β1 (TGF-β) or by tumor necrosis factor-α (TNF). The inhibition of the TGF-β-stimulated PG production appeared to reflect a diminished efficiency of arachidonic acid conversion into PGs by the cells, while the efficiency of substrate utilization for PG biosynthesis was unaffected by 1,25(OH)2D3 pretreatment in the unstimulated samples, or in samples stimulated with TNF or with TNF plus TGF-β. Free arachidonic acid levels were decreased following 1,25(OH)2D3 pretreatment in the TNF stimulated samples. hOB cell phospholipase A2 activity was measured in subcellular fractions, and this activity was decreased by 20-25% in the 1,25(OH)2D3 pretreated samples. The addition of the selective inhibitor AACOCF3 to the phospholipase A2 assays provided evidence that it was the cytoplasmic isoform of the enzyme that was affected by the 1,25(OH)2D3 pretreatment of the hOB cells. Thus, 1,25(OH)2D3 regulation of hOB cell biology includes significant effects on arachidonic acid metabolism. In turn, this could influence the effects of other hormones and cytokines whose actions include the stimulated production of bioactive arachidonic acid metabolites. J. Cell. Biochem. 68:237-246, 1998. © 1998 Wiley-Liss, Inc.
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  • 106
    ISSN: 0730-2312
    Keywords: TGF-β ; transcription factor ; rapid regulation ; tumor suppressor ; osteoblasts ; immunohistochemistry ; breast cancer stage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: This laboratory has previously identified a novel TGF-β inducible early gene (TIEG) in human osteoblasts [Subramaniam et al. (1995): Nucleic Acids Res 23:4907-4912]. Using TIEG specific polyclonal antibody and immunoprecipitation methods in normal human fetal osteoblast cells (hFOB cells), we have now demonstrated that TIEG encodes a 72-kDa protein whose levels are transiently increased at as early as 2 h of TGF-β treatment. Polarized confocal microscopic analysis of hFOB cells shows a nuclear localized TIEG protein in untreated cells under the conditions described under Methods. Interestingly, the levels of TIEG protein in the nuclei increase when the cells are treated with TGF-β1 for 2 h. In contrast, similar analyses of untreated human keratinocytes show a cytoplasmic localized TIEG protein that appears to be translocated to the nucleus after H2O2 treatment. Additional immunohistochemical studies have demonstrated that TIEG protein is expressed in epithelial cells of the placenta, breast, and pancreas, as well as in osteoblast cells of bone and selected other cells of the bone marrow and cerebellum with some cells showing a cytoplasmic localization and others a nuclear localization. All cells of the kidney display negative staining for this protein. Interestingly, a stage specific expression of TIEG protein is found in a dozen breast cancer biopsies, using immunohistochemistry. The cells in normal breast epithelium displays a high expression of TIEG protein, those in the in situ carcinoma display less than one-half of the levels, and those in the invasive carcinoma show a complete absence of the TIEG protein. TIEG has been localized to chromosome 8q22.2 locus, the same locus as the genes involved in osteopetrosis and acute myeloid leukemia and close to the c-myc gene locus and a locus of high polymorphism in cancer biopsies. The correlation between the levels of TIEG protein and the stage of breast cancer, its prime location in human chromosome 8q22.2, and past studies with pancreatic carcinoma, suggests that TIEG may play a role in tumor suppressor gene activities, apoptosis, or some other regulatory function of cell cycle regulation. J. Cell. Biochem. 68:226-236, 1998. © 1998 Wiley-Liss, Inc.
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  • 107
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 259-268 
    ISSN: 0730-2312
    Keywords: multifunctional Ca2+/calmodulin-dependent protein kinase ; cardiac isoforms ; muscle differentiation ; cell line Hgc2 ; adult rat heart ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Despite their important role in controlling the cardiac Ca2+ homeostasis, presence and functions of individual isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinase in the heart are not well studied. Here we report on expression of isoforms of the δ class in two differentiation states of the embryonic rat heart-derived cell line H9c2 compared to adult rat heart. Reverse transcription coupled polymerase chain reaction analysis revealed specific expression patterns of four variants of the δ class (δB, δC, δ4, δ9) in adult rat heart, H9c2 myoblasts, and skeletal muscle-like H9c2 myotubes. δC was identified as a common isoform with higher amounts in H9c2 cells and the prominent one in myoblasts. In contrast, expression of δ9 accompanied cardiac as well as skeletal muscle differentiation. Expression of δB, however, was representative for differentiated cardiac muscle, whereas δ4 expression coincided with differentiation into the skeletal muscle-like state. Our results demonstrate differentiation-dependent isoform expression of the δ class of the multifunctional Ca2+/calmodulin-dependent protein kinase of muscle. The identification of cardiac target proteins for this kinase, e.g. the α1-subunit of the L-type Ca2+ channel, the sarcoplasmic reticulum Ca2+-ATPase, phospholamban and the ryanodine receptor define H9c2 myoblasts as a suitable model system for further functional characterization of the identified cardiac δ isoforms. J. Cell. Biochem. 68:259-268, 1998. © 1998 Wiley-Liss, Inc.
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  • 108
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We are using viral oncogene probes to study the pathways by which osteoblast-specific gene expression is induced in ascorbic acid-treated MC3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directly to key cellular regulators and, as a result, represses tissue specific gene expression and blocks differentiation in a wide variety of cell types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family. The p300 family appears to be the primary target for E1A-mediated repression of tissue-specific gene expression in a variety of cell types. We have generated MC3T3-E1 cell lines that stably express either the wild-type 12S product or a mutant that targets p300/CBP, but not the pRB family. Using these constructs to dissect osteoblast differentiation, we found that targeting of p300/CBP appears to be sufficient to repress alkaline phosphatase expression, although a low but functional level of expression can be maintained if the pRB family is not targeted as well. Induction of alkaline phosphatase expression and activity can be dissociated from expression of late-stage markers such as osteocalcin and osteopontin. Surprisingly, cell lines exhibiting severe repression of alkaline phosphatase activity differentiate to a mineral-secreting phenotype much like normal MC3T3-E1 cells. Osteopontin induction is dependent on at least a minimal level of alkaline phosphatase activity, although it is not dependent on induction of alkaline phosphatase at the RNA level. If alkaline phosphatase is supplied exogenously, osteopontin expression can be induced in conditions in which endogenous alkaline phosphatase is severely repressed. J. Cell. Biochem. 68:269-280, 1998. © 1998 Wiley-Liss, Inc.
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  • 109
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 281-285 
    ISSN: 0730-2312
    Keywords: QM ; large P-antigen ; 60S ribosomal subunit ; colocalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: QM is a human cDNA originally isolated as a transcript elevated in a nontumorigenic Wilms' tumor microcell hybrid, relative to the tumorigenic parental cell line. The QM gene encodes a 24 kDa basic protein that peripherally associates with the ribosomes. Recently, the gene for this protein has also been shown in Saccharomyces cerevisiaeto encode an essential 60S ribosomal subunit protein that is required for the joining of the 40S and 60S subunits. Since the association of QM with ribosomes can be disrupted with 1M NaCl, which has no effect on the association of core ribosomal proteins, indirect immunofluorescent cell staining was performed to colocalize the QM protein with the human large P-antigen, a core ribosomal protein of the 60S subunit, and to determine whether the assembly of the QM protein onto the 60S ribosomal subunit occurs in the nucleolus or in the cytoplasm. Our results reveal that QM co-localizes with the large P-antigen only to the cytoplasm where the rough endoplasmic reticulum is found and not to the nucleolus where ribosome assembly occurs. This finding suggests that the QM protein is most likely involved in a late step of the 60S subunit assembly and is added to the 60S ribosomal subunit in the cytoplasm and not in the nucleolus. J. Cell. Biochem. 68:281-285, 1998. © 1998 Wiley-Liss, Inc.
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  • 110
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 309-327 
    ISSN: 0730-2312
    Keywords: in vitro replication ; ors8 ; Oct-1 transcription factor ; POU domain ; mammalian autonomously replicating DNA sequence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A 186-base pair fragment of ors8, a mammalian autonomously replicating DNA sequence isolated by extrusion of nascent monkey DNA in early S phase, has previously been identified as the minimal sequence required for replication function in vitro and in vivo. This 186-base pair fragment contains, among other sequence characteristics, an imperfect consensus binding site for the ubiquitous transcription factor Oct-1. We have investigated the role of Oct-1 protein in the in vitro replication of this mammalian origin. Depletion of the endogenous Oct-1 protein, by inclusion of an oligonucleotide comprising the Oct-1 binding site, inhibited the in vitro replication of p186 to approximately 15-20% of the control, whereas a mutated Oct-1 and a nonspecific oligonucleotide had no effect. Furthermore, immunodepletion of the Oct-1 protein from the HeLa cell extracts by addition of an anti-POU antibody to the in vitro replication reactioninhibited p186 replication to 25% of control levels. This inhibition of replication could be partially reversed to 50-65% of control levels, a two- to threefold increase, upon the addition of exogenous Oct-1 POU domain protein.Site-directed mutagenesis of the octamer binding site in p186 resulted in a mutant clone, p186-MutOct, which abolished Oct-1 binding but was still able to replicate as efficiently as the wild-type p186. The results suggest that Oct-1 protein is an enhancing component in the in vitro replication of p186 but that its effect on replication is not caused through direct binding to the octamer motif. J. Cell. Biochem. 68:309-327, 1998. © 1998 Wiley-Liss, Inc.
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  • 111
    ISSN: 0730-2312
    Keywords: PEPCK ; adipocytes ; transcription ; fatty acids ; fibrates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Phosphoenolpyruvate carboxykinase (PEPCK) exerts a glyceroneogenic function in adipocytes in which transcription of its gene is increased by unsaturated fatty acids and fibrates. We used cultured rat adipose tissue fragments and 3T3-F442A adipocytes to show that the antidiabetic thiazolidinedione BRL 49653, a ligand and an activator of the γ isoform of peroxisome proliferator activated receptors (PPARγ), is a potent inducer of PEPCK mRNA. In 3T3-F442A adipocytes, the effect of BRL 49653 is rapid and concentration dependent, with a maximum reached at 1 μM and a half-maximum at 10-100 nM. PEPCK mRNA is similarly induced by the natural ligand of PPARγ, the 15-deoxy-Δ12-14 prostaglandin J2. These observations strongly suggest that PPARγ is a primary regulator of PEPCK gene expression in adipocytes. Dexamethasone at 10 nM repress induction of PEPCK mRNA by 1 μM BRL 49653, 0.32 mM oleate, or 1 mM clofibrate, in a cycloheximide-independent manner. The antiglucocorticoid RU 38486 prevents dexamethasone action, demonstrating involvement of the glucocorticoid receptor. Stable transfectants of 3T3-F442A adipocytes bearing -2100 to +69 base pairs of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene respond to 1 μM BRL 49653 or 1 mM clofibrate by a large increase in CAT activity, which is prevented by the simultaneous addition of 10 nM dexamethasone. Hence, in adipocytes, glucocorticoids act directly through the 5′-flanking region of the PEPCK gene to repress, in a dominant fashion, the stimulation of PEPCK gene transcription by thiazolidinediones and fibrates. J. Cell. Biochem. 68:298-308, 1998. © 1998 Wiley-Liss, Inc.
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  • 112
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 287-297 
    ISSN: 0730-2312
    Keywords: aorta ; mineralization ; calcification ; hydroxyapatite ; inhibitors ; arteriosclerosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na2HPO4 and 0.1 mM CaCl2 were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27-29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis. J. Cell. Biochem. 68:287-297, 1998. © 1998 Wiley-Liss, Inc.
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  • 113
    ISSN: 0730-2312
    Keywords: cell proliferation ; tumor progression ; EGF receptor ; ErbB ; HER1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an activating ligand for the EGF receptor (HER1/ErbB1) and the high-affinity receptor for diphtheria toxin (DT) in its transmembrane form (proHB-EGF). HB-EGF was immunolocalized within human benign and malignant prostatic tissues, using monospecific antibodies directed against the mature protein and against the cytoplasmic domain of proHB-EGF. Prostate carcinoma cells, normal glandular epithelial cells, undifferentiated fibroblasts, and inflammatory cells were not decorated by the anti-HB-EGF antibodies; however, interstitial and vascular smooth muscle cells were highly reactive, indicating that the smooth muscle compartments are the major sites of synthesis and localization of HB-EGF within the prostate. In marked contrast to prostatic epithelium, proHB-EGF was immunolocalized to seminal vesicle epithelium, indicating differential regulation of HB-EGF synthesis within various epithelia of the reproductive tract. HB-EGF was not overexpressed in this series of cancer tissues, in comparison to the benign tissues. In experiments with LNCaP human prostate carcinoma cells, HB-EGF was similar in potency to epidermal growth factor (EGF) in stimulating cell growth. Exogenous HB-EGF and EGF each activated HER1 and HER3 receptor tyrosine kinases and induced tyrosine phosphorylation of cellular proteins to a similar extent. LNCaP cells expressed detectable but low levels of HB-EGF mRNA; however, proHB-EGF was detected at the cell surface indirectly by demonstration of specific sensitivity to DT. HB-EGF is the first HER1 ligand to be identified predominantly as a smooth muscle cell product in the human prostate. Further, the observation that HB-EGF is similar to EGF in mitogenic potency for human prostate carcinoma cells suggests that it may be one of the hypothesized stromal mediators of prostate cancer growth. J. Cell. Biochem. 68:328-338, 1998. © 1998 Wiley-Liss, Inc.
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  • 114
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 339-354 
    ISSN: 0730-2312
    Keywords: glutathione ; reactive oxygen intermediates ; HIV ; signal transduction ; cytokines ; redox state ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α). While virtually every cell responds to TNF-α with gene activation, the extent of TNF-α-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-α membrane interaction, are part of this TNF-α-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-α. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-α challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV- normal fibroblasts. In contrast, following TNF-α challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-α-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-α challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-α, suggest a biochemical rationale for the recognized TNF-α AIDS-KS clinical correlation. J. Cell. Biochem. 68:339-354, 1998. © 1998 Wiley-Liss, Inc.
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  • 115
    ISSN: 0730-2312
    Keywords: mechanical loading ; gene expression ; osteopontin ; myeloperoxidase ; rats ; differential display ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function. J. Cell. Biochem. 68:355-365, 1998. © 1998 Wiley-Liss, Inc.
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  • 116
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    Journal of Cellular Biochemistry 68 (1998), S. 366-377 
    ISSN: 0730-2312
    Keywords: PC-1 ; insulin action ; insulin resistance ; insulin receptor ; tyrosine kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: An elevated content of membrane glycoprotein PC-1 has been observed in cells and tissues of insulin resistant patients. In addition, in vitro overexpression of PC-1 in cultured cells induces insulin resistance associated with diminished insulin receptor tyrosine kinase activity. We now find that PC-1 overexpression also influences insulin receptor signaling at a step downstream of insulin receptor tyrosine kinase, independent of insulin receptor tyrosine kinase. In the present studies, we employed Chinese hamster ovary cells that overexpress the human insulin receptor (CHO IR cells; ∼106 receptors per cell), and transfected them with human PC-1 c-DNA (CHO IR PC-1). In CHO IR PC-1 cells, insulin receptor tyrosine kinase activity was unchanged, following insulin treatment of cells. However, several biological effects of insulin, including glucose and amino acid uptake, were decreased. In CHO IR PC-1 cells, insulin stimulation of mitogen-activated protein (MAP) kinase activity was normal, suggesting that PC-1 overexpression did not affect insulin receptor activation of Ras, which is upstream of MAP kinase. Also, insulin-stimulated phosphatidylinositol (PI)-3-kinase activity was normal, suggesting that PC-1 overexpression did not interfere with the activation of this enzyme by insulin receptor substrate-1. In these cells, however, insulin stimulation of p70 ribosomal S6 kinase activity was diminished. These studies suggest, therefore, that, in addition to blocking insulin receptor tyrosine kinase activation, PC-1 can also block insulin receptor signaling at a post-receptor site. J. Cell. Biochem. 68:366-377, 1998. © 1998 Wiley-Liss, Inc.
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    Journal of Cellular Biochemistry 68 (1998), S. 378-388 
    ISSN: 0730-2312
    Keywords: apoptosis ; growth suppression ; retinoic acid receptors ; ovarian cancer ; AHPN ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have used conformationally restricted retinoids to investigate the role of individual RAR subtypes and RXR in mediating the growth response of ovarian tumor cells to retinoids. Our results show that treatment of all-trans-RA-sensitive CAOV-3 cells with retinoids that bind and activate a single RAR or RXR led to a partial inhibition of growth. Treatment of all-trans-RA- resistant SKOV-3 cells did not alter growth. Maximum inhibition of growth, comparable to that observed following treatment with natural retinoids such as all-trans-RA and 9-cis-RA, was obtained only following treatment with a combination of an RAR-selective compound and an RXR-selective one. These results suggest that activation of both RAR and RXR classes is required in order to obtain maximum inhibition of ovarian tumor cell growth by retinoids. In addition, one compound, AHPN, was found to inhibit both RA-sensitive CAOV-3 and RA-resistant SKOV-3 cells. Further study of the effects of this retinoid showed that AHPN acts through an apoptotic pathway. Taken together, our results suggest that retinoids may serve as effective anti-proliferative agents in the treatment of ovarian cancer. J. Cell. Biochem. 68:378-388, 1998. © 1998 Wiley-Liss, Inc.
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  • 118
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    Journal of Cellular Biochemistry 68 (1998), S. 389-401 
    ISSN: 0730-2312
    Keywords: cytoskeleton ; cell motility ; intracellular dynamics ; stress fibers ; heavy chain ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may be performed. Non-muscle myosin IIA was purified from bovine platelets, non-muscle myosin IIB from bovine brain, and smooth muscle myosin II from turkey gizzards. After being fluorescently labeled with tetramethylrhodamine-5-iodoacetamide or with a succinimidyl ester of cy5, they retained the following properties: (1) reversible assembly into thick filaments, (2) actin-activatable MgATPase, (3) phosphorylation by myosin light chain kinase, (4) increased MgATPase upon light-chain phosphorylation, (5) interconversion between 6S and 10S conformations, and (6) distribution into endogenous myosin II-containing structures when microinjected into cultured cells. These fluorescent analogues can be used to visualize isoform-specific dynamics of myosin II in living cells. J. Cell. Biochem. 68:389-401, 1998. © 1998 Wiley-Liss, Inc.
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  • 119
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    Journal of Cellular Biochemistry 68 (1998), S. 403-410 
    ISSN: 0730-2312
    Keywords: extracellular matrix ; gene therapy ; collagen ; matrix metalloproteinase ; tissue inhibitor of metalloproteinase ; transforming growth factor ; decorin ; cardiomyopathy ; hypertrophy ; ischemia ; fibrosis; functional genomics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure.There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403-410, 1998. © 1998 Wiley-Liss, Inc.
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  • 120
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    Journal of Cellular Biochemistry 68 (1998), S. 411-426 
    ISSN: 0730-2312
    Keywords: bone marrow stroma ; human ; differentiation ; TGF-β ; BMP-2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β2) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β2 increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β2 regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β2 led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β2 attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β2-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β2 on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β2 response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β2 did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β2 acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β2 and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411-426, 1998. © 1998 Wiley-Liss, Inc.
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  • 121
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    Journal of Cellular Biochemistry 68 (1998), S. 427-435 
    ISSN: 0730-2312
    Keywords: α2-macroglobulin ; albumin ; placenta ; zinc ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have investigated the binding and internalization of α2-macroglobulin and serum albumin by human placental syncytiotrophoblast cells in vitro. The time course (obtained at 4°C) of α2-macroglobulin binding indicated that an equilibrium was reached after 4 h. The binding of 125I-labelled α2-macroglobulin to syncytiotrophoblast cells was competitively reduced in the presence of excess unlabelled α2-macroglobulin. When the concentration-dependence of binding was examined over a wide concentration range, non-linear regression analysis yielded a Kd of 6.4 nM. In the case of albumin, binding was weak and ligand dissociated from the cell surface during aqueous washing making it impractical to analyze the binding reaction. In other experiments, syncytiotrophoblast cells were incubated with 125I-labelled α2-macroglobulin at 37°C. Under these conditions, trypsin-resistant cell-associated radioactivity increased with time consistent with ligand internalization. 125I-Labelled-ligand was internalized with a t1/2 of about 5 min. After a lag period some radioactivity was released back into the incubation medium. When measured at times up to 210 min, this was found to consist of mostly TCA-precipitable material that had been lost from the cell surface. However, when the incubation was extended to 24 h, almost 15% of the initial cell-associated radioactivity was released to the extracellular medium as TCA-soluble material, consistent with a slow rate of ligand degradation. The specific binding of 65Zn-labelled α2M was similar to that of the 125I-labelled ligand and trypsin-resistance measurements provided evidence of α2M-mediated 65Zn uptake. These results support a role for syncytiotrophoblast in the metabolism of α2-macroglobulin during pregnancy and are also consistent with a role for α2-macroglobulin in the maternal-fetal transport of zinc. J. Cell. Biochem. 68:427-435, 1998. © 1998 Wiley-Liss, Inc.
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  • 122
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    Journal of Cellular Biochemistry 68 (1998), S. 436-445 
    ISSN: 0730-2312
    Keywords: mouse ; PDI family proteins ; retinoic acid ; dibutyryl cAMP ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be co-immunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation. J. Cell. Biochem. 68:436-445, 1998. © 1998 Wiley-Liss, Inc.
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  • 123
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    Journal of Cellular Biochemistry 68 (1998), S. 446-456 
    ISSN: 0730-2312
    Keywords: IGF-I ; IGF-II ; cAMP ; PKA ; PKC ; prostaglandin ; osteoblasts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone cells synthesize and respond to IGF-I and IGF-II which contribute to bone remodeling and linear growth. In osteoblasts, prostaglandin (PG)E2 stimulates IGF-I but not IGF-II synthesis through a cAMP-dependent protein kinase A (PKA)-related event. However, protein kinase C (PKC) activation by PGE2 enhances replication and protein synthesis by less differentiated periosteal cells more so than in osteoblast-enriched cultures from fetal rat bone. Using various PGs and other PKA and PKC pathway activators, the importance of these aspects of PGE2 activity has now been examined on IGF receptors in these bone cell culture models. PGE2 and other agents that activate PKA enhanced 125I-IGF-II binding to type 2 IGF receptors on both cell populations. In contrast, agents that activate PKC enhanced 125I-IGF-I binding to type 1 receptors on less differentiated bone cells, and of these, only phorbol myristate acetate (PMA), which activates PKC in a receptor-independent way, was effective in osteoblast-enriched cultures. No stimulator increased total type 1 receptor protein in either cell population. Consequently, ligand binding to type 1 and type 2 IGF receptors is differentially modulated by specific intracellular pathways in bone cells. Importantly, changes in apparent type 1 receptor number occur rapidly and may do so at least in part through post-translational effects. These results may help to predict new ways to manipulate autocrine or paracrine actions by IGFs in skeletal tissue. J. Cell. Biochem. 68:446-456, 1998. © 1998 Wiley-Liss, Inc.
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  • 124
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    Journal of Cellular Biochemistry 68 (1998), S. 457-471 
    ISSN: 0730-2312
    Keywords: coated vesicles ; acetylcholine receptors ; AP180 ; myotube ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Monoclonal antibodies were generated to vesicular membranes of clathrin coated vesicles enriched for acetylcholinesterase (AChE). One of these, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunoblots of muscle extracts and brain purified clathrin coated vesicles show that C172 recognizes a 100 kd band in muscle, but a 180 kd band in brain. Western blots of purified AP180 protein stained with the two antibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to known sequences of AP180 were used to map the epitope for C172 within the brain AP180 sequence. On immunoblots of digested AP180, all AP180 antibodies and C172 recognized a 100 kd tryptic fragment, however only C172 recognized a smaller 60 kd. Our results suggest that the C172 epitope is located within amino acids 305-598 of the AP180 sequence. Confocal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distinct from that observed when cells are stained with γ adaptin antibody which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quantitative analysis of C172 reactive vesicles revealed a significant increase in number of vesicles present around the nuclei associated with the acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with homology to the neuron-specific coated vesicle protein AP180, is present in muscle cells associated with vesicles showing significant concentration around postsynaptic nuclei present in close proximity to AChR clusters. J. Cell. Biochem. 68:457-471, 1998. © 1998 Wiley-Liss, Inc.
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  • 125
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    Keywords: taxol ; microtubules ; vimentin ; intermediate filaments ; protein phosphorylation ; protein kinases ; inhibitors ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Taxol, a microtubule stabilizing agent, has been extensively investigated for its antitumor activity. The cytotoxic effect of taxol is generally attributed to its antimicrotubule activity and is believed to be cell cycle dependent. Herein, we report that taxol induces hyperphosphorylation and reorganization of the vimentin intermediate filament in 9L rat brain tumor cells, in concentration- and time-dependent manner. Phosphorylation of vimentin was maximum at 10-6 M of taxol treatment for 8 h and diminished at higher (10-5 M) concentration. Enhanced phosphorylation of vimentin was detectable at 2 h treatment with 10-6 M taxol and was maximum after 12 h of treatment. Taxol-induced phosphorylation of vimentin was largely abolished in cells pretreated with staurosporine and bisindolymaleimide but was unaffected by H-89, KT-5926, SB203580, genistein, and olomoucine. Thus, protein kinase C may be involved in this process. Hyperphosphorylation of vimentin was accompanied by rounding up of cells as revealed by scanning electron microscopy. Moreover, there was a concomitant reorganization of the vimentin intermediate filament in the taxol-treated cells, whereas the microtubules and the actin microfilaments were less affected. Taken together, our data demonstrate that taxol induces hyperphosphorylation of vimentin with concomitant reorganization of the vimentin intermediate filament and that this process may be mediated via a protein kinase C signaling pathway. J. Cell Biochem. 68:472-483, 1998. © 1998 Wiley-Liss, Inc.
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  • 126
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    Journal of Cellular Biochemistry 68 (1998), S. 484-499 
    ISSN: 0730-2312
    Keywords: YY1 ; zinc finger ; high-molecular-weight complex ; plasmid transfection ; nuclear matrix association ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: YY1 is a zinc finger-containing transcription factor that can both repress and activate transcription. YY1 appears to use multiple mechanisms to carry out its diverse functions. Recently, it was observed that YY1 can exist in multiple nuclear compartments. In addition to being present in the nuclear extract fraction, YY1 is also a component of the nuclear matrix. We show that YY1 can be sequestered in vivo into a high-molecular-weight complex and can be dislodged from this complex either by treatment with formamide or by incubation with an oligonucleotide containing the YY1 DNA binding site sequence. By transfecting plasmids expressing various YY1 deletion constructs and subsequent nuclear fractionation, we have identified sequences necessary for association with the nuclear matrix. These sequences (residues 256-340) co-localized with those necessary for in vivo sequestration of YY1 into the high-molecular-weight complex. We have also characterized YY1 sequences necessary for repression of activated transcription (residues 333-371) and those necessary for masking of the YY1 transactivation domain (residues 371-397). Sequences that repress activated transcription partially overlap YY1 sequences necessary for association with the nuclear matrix. However, these sequences are distinct from those that appear to mask the YY1 transactivation domain. The potential role of nuclear matrix association in controlling YY1 function is discussed. J. Cell. Biochem. 68:484-499, 1998. © 1998 Wiley-Liss, Inc.
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  • 127
    ISSN: 0730-2312
    Keywords: transcription factor ; nuclear matrix ; YY1 ; amino acids ; functional regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The multifunctional transcription factor YY1 is associated with the nuclear matrix. In osteoblasts, the interaction of several nuclear matrix-associated transcription factors with the bone specific osteocalcin gene contributes to tissue-specific and steroid hormone-mediated transcription. A canonical nuclear matrix targeting signal (NMTS) is present in all members of the AML/CBFβ transcription factor family, but not in other transcription factors. Therefore, we defined sequences that direct YY1 (414 amino acids) to the nuclear matrix. A series of epitope tagged deletion constructs were expressed in HeLa S3 and in human Saos-2 osteosarcoma cells. Subcellular distribution was determined in whole cells and nuclear matrices in situ by immunofluorescence. We demonstrated that amino acids 257-341 in the C-terminal domain of YY1 are necessary for nuclear matrix association. We also observed that sequences within the N-terminal domain of YY1 permit weak nuclear matrix binding. Our data further suggest that the Gal4 epitope tag contains sequences that affect subcellular localization, but not targeting to the nuclear matrix. The targeted association of YY1 with the nuclear matrix provides an additional level of functional regulation for this transcription factor that can exhibit positive and negative control. J. Cell. Biochem. 68:500-510, 1998. © 1998 Wiley-Liss, Inc.
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  • 128
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    Journal of Cellular Biochemistry 68 (1998), S. 511-524 
    ISSN: 0730-2312
    Keywords: actin ; permeability ; reoxygenation ; signal transduction ; cytoskeletal rearrangement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Hypoxia/reoxygenation injury to cultured endothelial cells results in cytoskeletal rearrangement and second messenger activation related to increased monolayer junctional permeability. Cytoskeletal rearrangement by reactive oxygen species may be related to specific activation of the phospholipase D (PLD) pathway. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 μM) or metabolites of the PLD pathway for 1-60 min. Changes in cAMP levels, Ca2+ levels, PIP2 production, filamin distribution, and intercellular gap formation are then quantitated. H2O2-induced filamin translocation from the membrane to the cytosol occurs after 1-min H2O2 treatment, while intercellular gap formation significantly increases after 15 min. H2O2 and phosphatidic acid exposure rapidly decrease intracellular cAMP levels, while increasing PIP2 levels in a Ca2+-independent manner. H2O2-induced cAMP decreases are prevented by inhibiting phospholipase D. H2O2-induced cytoskeletal changes are prevented by inhibiting phospholipase D, phosphatidylinositol-4-phosphate kinase, phosphoinositide turnover, or by adding a synthetic peptide that binds PIP2. These data indicate that metabolites produced downstream of H2O2-induced PLD activation may mediate filamin redistribution and F-actin rearrangement. J. Cell. Biochem. 68:511-524, 1998. © 1998 Wiley-Liss, Inc.
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  • 129
    ISSN: 0730-2312
    Keywords: nuclear matrix ; replication origin ; topoisomerase II-mediated DNA loop excision ; DNA loop anchorage sites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions. J. Cell. Biochem. 69:13-18, 1998. © 1998 Wiley-Liss, Inc.
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    Journal of Cellular Biochemistry 69 (1998), S. 1-12 
    ISSN: 0730-2312
    Keywords: two-hybrid system ; vitamin D receptor ; retinoid X receptor ; vitamin D ; protein L7 ; basic region leucine zipper domain ; coregulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes. Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D. Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR. Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937. Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast. We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element. Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid. Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR. Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements. J. Cell. Biochem. 69:1-12, 1998. © 1998 Wiley-Liss, Inc.
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    Journal of Cellular Biochemistry 69 (1998), S. 19-29 
    ISSN: 0730-2312
    Keywords: interleukin-1 ; reactive oxygen species ; nitric oxide ; c-fos ; collagenase ; chondrocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interleukin-1β (IL-1) is implicated in cartilage destruction in arthritis through promotion of matrix metalloproteinase production. Upregulation of collagenase gene expression by IL-1 is known to require the transactivators Fos and Jun. Recently, reactive oxygen species (ROS) have been suggested to act as intracellular signaling molecules mediating the biological effects of cytokines. Here, we demonstrated ROS production by IL-1-stimulated bovine chondrocytes and that neutralizing ROS activity by the potent antioxidant, N-acetylcysteine, or inhibiting endogenous ROS production by diphenyleneiodonium (DPI), significantly attenuated IL-1-induced c-fos and collagenase gene expression. The inhibitory effect of DPI implicates enzymes such as NADPH oxidase in the endogenous production of ROS. Chondrocytes were also found to produce nitric oxide (NO) upon IL-1 stimulation. That NO may mediate part of the inducing effects of IL-1 was supported by the observation that L-NG-monomethylarginine, a NO synthase inhibitor, partially inhibited IL-1-regulated collagenase expression. Moreover, treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, was sufficient to induce collagenase mRNA levels. In summary, our results suggest that ROS released in response to IL-1 may function as second messengers transducing extracellular stimuli to their targets in the nucleus, leading to augmentation of gene expression. J. Cell. Biochem. 69:19-29, 1998. © 1998 Wiley-Liss, Inc.
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  • 132
    ISSN: 0730-2312
    Keywords: genistein ; breast cancer ; p21WAF1/CIP1 ; G2/M arrest ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Genistein has been proposed to be responsible for lowering the rate of breast cancer in Asian women but the mechanism for this chemopreventive effect in vivo is unknown. In this study, we present in vitro evidence that genistein inhibits cell proliferation similarly in ER-positive and ER-negative human breast carcinoma cell lines. This inhibition is associated with specific G2/M arrest and induction of p21WAF1/CIP1 expression. Genistein results in a five- to six-fold increase in p21WAF1/CIP1 mRNA levels and a three- to four-fold increase in protein levels, only a 1.5-fold increase in p21WAF1/CIP1 transcription but a three- to six-fold increase in p21WAF1/CIP1 mRNA stability. The increase in p21WAF1/CIP1 is followed by increased apoptosis. The similar effects of genistein on a number of breast carcinoma cell lines with different ER and p53 status suggest that the actions of genistein reported here are mediated through ER and p53 independent mechanisms. The chemopreventive effects of genistein in vivo could be mediated along an identical or similar anti-proliferative pathway. J. Cell. Biochem. 69:44-54, 1998. © 1998 Wiley-Liss, Inc.
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    Journal of Cellular Biochemistry 69 (1998), S. 30-43 
    ISSN: 0730-2312
    Keywords: hyperthermia ; calreticulin ; chaperone complexes ; prompt glycosylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Acute heat stress leads to the glycosylation of a “prompt” stress glycoprotein, P-SG67/64, identified as calreticulin. In the present study, we used immunoprecipitation to investigate the interactions of P-SG/calreticulin with other proteins during cellular recovery from heat stress. In heat-stressed CHO and M21 cells, both glycosylated and unglycosylated P-SGs interact with HSP90, GRP94, GRP78, and the other prompt stress glycoprotein, P-SG50, in an ATP-independent manner. Specificity of HSP-P-SG interactions was determined by chemical cross-linking with the homo-bifunctional agent DSP (3,3′-dithiobis[succinimidyl propionate]). Characterization of the cross-linked complexes involving calreticulin and heat shock proteins (HSPs) showed an average mass of 400-600 kDa by gel filtration chromatography. Overall, the consistent association of glycosylated and unglycosylated calreticulin with P-SG50 and unglycosylated HSPs suggests that P-SG/calreticulin is an active member of the cast of glycone/aglycone chaperones that cooperate to achieve cellular recovery from acute heat stress. J. Cell. Biochem. 69:30-43, 1998. © 1998 Wiley-Liss, Inc.
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  • 134
    ISSN: 0730-2312
    Keywords: TGF-α ; antisense oligonucleotides ; head and neck cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interruption of an autocrine growth pathway involving TGF-α and EGFR may inhibit tumor growth and improve survival in head and neck cancer patients. We previously demonstrated that biopsy specimens and established cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress TGF-α and its receptor, epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies showed that TGF-α and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting a role for this ligand-receptor pair in an autocrine growth pathway. To confirm that TGF-α contributes to autocrine growth, we examined the effect of down regulation of TGF-α protein on SCCHN cell proliferation. Treatment of 6 SCCHN cell lines with antisense oligodeoxynucleotides targeting the translation start site of human TGF-α mRNA decreased TGF-α protein production by up to 93% and reduced cell proliferation by a mean of 76.2% compared to a 9.7% reduction with sense oligonucleotide (range P〈0R 〉 = 0.036-0.0001). TGF-α antisense oligonucleotide exposure also decreased TGF-α protein levels in normal oropharyngeal mucosal epithelial cells, however their growth rate was not affected. These findings indicate that TGF-α is participating in an autocrine signaling pathway in transformed, but not in normal mucosal epithelial cells, that promotes proliferation. J. Cell. Biochem. 69:55-62, 1998. © 1998 Wiley-Liss, Inc.
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  • 135
    ISSN: 0730-2312
    Keywords: angiotensin II ; G proteins ; Src tyrosine kinases ; c-Fos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Angiotensin II stimulates a biphasic activation of Raf-1, MEK, and ERK in WB liver epithelial cells. The first peak of activity is rapid and transient and is followed by a sustained phase. Angiotensin II also causes a rapid activation of p21ras in these cells. Moreover, two Src family kinases (Fyn and Yes) were activated by angiotensin II in a time- and concentration-dependent manner. Microinjection of antibodies against Fyn and Yes blocked angiotensin II-induced DNA synthesis and c-Fos expression in WB cells, indicating an obligatory involvement of these tyrosine kinases in the activation of the ERK cascade by angiotensin II. Finally, substantial reduction of the angiotensin II-stimulated activation of Fyn, Raf-1, ERK, and expression of c-Fos by pertussis toxin pretreatment argues that G proteins of the Gi family as well as the Gq family are involved in angiotensin II-mediated mitogenic pathways in WB cells. J. Cell. Biochem. 69:63-71, 1998. © 1998 Wiley-Liss, Inc.
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  • 136
    ISSN: 0730-2312
    Keywords: human oocytes ; immunogold labeling ; splicing factors ; coilin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The distribution of two splicing components (snRNP and SC-35) and coilin were studied by immunogold/electron microscopy in human oocytes from antral follicles at different levels of transcriptional activity (i.e., active, intermediate, and inactive). The results showed a decrease of snRNPs and SC-35 in the karyoplasm as the oocytes progress from a transcriptionally active to the inactive state. The main areas of accumulation of both these splicing components in all stages of oocytes appeared to be the interchromatin granule clusters (IGCs). Within the IGCs, the two splicing components seemed to be spatially segregated, with the snRNPs predominantly bound to the fibrillar region, whereas the SC-35 factors are being enriched in the granular zone. The p80 coilin was found only in the nucleolus-like body (NLB), which is present in all three stages of oocytes; no coiled bodies were evident. These data are consistent with the notion that splicing occurs in the karyoplasm and that the splicing components are mobilized from a storage site (IGCs) to the site of action. J. Cell. Biochem. 69:72-80, 1998. © 1998 Wiley-Liss, Inc.
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  • 137
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    Journal of Cellular Biochemistry 69 (1998), S. 81-86 
    ISSN: 0730-2312
    Keywords: cell communication ; osteoblasts ; stromal cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We characterized the formation and regulation of the gap junction in calvarial osteoblasts and in a series of subtypes from marrow stromal cells. The stromal cells included osteogenic, chondro-osteogenic, and endothelial cells. The cell coupling was measured by using fluorescence dye injected into single cells, and its migration to neighboring cells was measured. The functional coupling of cells was highly expressed by the osteoblastic cells. This process is mediated through fast changes in intracellular Ca+2 levels. Calcium ionophore (A 23187) demonstrated an uncoupling effect on the cells. In addition, the exposure of the cells to the parathyroid hormone increased the formation of the gap junction complex; the highest level was demonstrated in the osteoblastic cells. J. Cell. Biochem. 69:81-86, 1998. © 1998 Wiley-Liss, Inc.
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  • 138
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    Journal of Cellular Biochemistry 69 (1998), S. 87-93 
    ISSN: 0730-2312
    Keywords: MAP kinase pathways ; JNK ; human osteoblasts ; interleukin-1β ; UMR-106 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We recently demonstrated the activation of extracellular signal- regulated protein kinase 1 and 2 (ERK1 and ERK2) by IGF-1, FGF-2, and PDGF-BB in normal human osteoblastic (HOB) cells as well as in rat and mouse osteoblastic cells. In this report, we have examined whether c-Jun NH2-Terminal Kinase (JNK) pathway is activated by growth factors and interleukin-1β (IL-1β) in normal HOB and rat UMR-106 cells using immune-complex kinase assay and anti-active JNK antibody, which recognizes activated forms of both JNK1 and JNK2. Results have demonstrated the presence of JNK1 and JNK2 proteins in normal HOB and UMR-106 cells. Both JNK1 and JNK2 were activated by IL-1β. IL-1β preferentially activated JNK pathway in a dose- and time-dependent manner and had little effect on ERK pathway. On the other hand, FGF-2 did not activate JNK but most strongly activated ERK pathway. The activation of JNK was maximal at 20 min whereas maximal activation of ERK1 and ERK2 was observed within 10 min. Results have clearly demonstrated that IL-1β preferentially activates JNK pathway whereas FGF-2 activates ERK pathway in normal human and rat UMR-106 osteoblastic cells. J. Cell. Biochem. 69:87-93, 1998. © 1998 Wiley-Liss, Inc.
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  • 139
    ISSN: 0730-2312
    Keywords: mechanical strain ; interleukin (IL)-α and β gene expression ; proliferation ; protein synthesis ; morphology ; keratinocyte biology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recent studies in our laboratory have demonstrated that mechanical strain alters many facets of keratinocyte biology including proliferation, protein synthesis, and morphology. IL-1 is known to play an important role in the autocrine regulation of these basic cellular properties under basal and stimulated conditions. However, it is not known whether IL-1 plays a role in strain-induced alteration of keratinocyte biology. Thus, the objective of this study was to test the hypothesis that cyclic strain stimulates IL-1 expression and that strain-induced changes in keratinocyte function is regulated by IL-1. To test this hypothesis, we examined the effect of cyclic strain (10% average deformation) on keratinocyte IL-1 gene expression and the effect of neutralizing antibodies of IL-1α and IL-1β on strain-induced changes in keratinocyte proliferation, morphology, and orientation. Northern blot analyses demonstrated that steady state levels of IL-1α and β mRNA were elevated by 4 h, peaked at 12 h of cyclic strain (IL-1α, 304 ± 14.2%; IL-1β, 212 ± 5.6% increase vs. static controls) and decreased gradually by 24 h. IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.01 μg/ml) significantly blocked strain-induced keratinocyte proliferation as well as the basal rate of proliferation. In contrast, IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.1 μg/ml) had no effect on strain-induced morphological changes such as elongation and alignment. We conclude that mechanical strain induces IL-1 mRNA expression in keratinocytes. The role of IL-1 in mediating strain-induced changes in keratinocyte biology remains to be determined but appears to be independent of morphological changes. J. Cell. Biochem. 69:95-103, 1998. © 1998 Wiley-Liss, Inc.
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  • 140
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    Journal of Cellular Biochemistry 69 (1998), S. 104-116 
    ISSN: 0730-2312
    Keywords: mRNA export ; cell cycle ; gene transfection ; cultured mammalian cells ; hnRNP L ; nuclear transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human β-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless β-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes. J. Cell. Biochem. 69:104-116, 1998. © 1998 Wiley-Liss, Inc.
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  • 141
    ISSN: 0730-2312
    Keywords: chromosome architecture ; disassembly ; reassembly ; proteases ; in vitro model ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Topoisomerase II has been suggested to play a major role in chromosome organization based on its DNA decatenating activity and its ability to mediate direct binding interactions between DNA and nuclear matrix. However, this latter point remains controversial. Here we address the question of whether the chromatin binding activity of Topoisomerase II is sufficient to modify chromosome form using whole mammalian chromosomes in vitro. Intact chromosomes were microsurgically removed from living cells and disassembled by treatment with protease or heparin. When these disassembled chromosomes were incubated with recombinant human Topoisomerase II, the enzyme became incorporated into chromatin and reassembly resulted, leading to almost complete restoration of pre-existing chromosome shape and position within minutes. Chromosome reconstituition by Topoisomerase II was dose-dependent, saturable, and appeared to be controlled stoichiometrically, rather than enzymatically. Similar reassembly was observed in the absence of ATP and when a catalytically inactive thermosensitive Topoisomerase II mutant was used at the restrictive temperature. Chromosome recondensation also could be induced after the strand-passing activity of Topoisomerase II was blocked by treatment with an inhibitor of its catalytic activity, amsacrine. When a non-hydrolyzable β,γ-imido analog of ATP (AMP-PNP) was used to physiologically fix bound Topoisomerase II enzyme in a closed form around DNA, subsequent chromosome disassembly was prevented in the presence of high salt. These data suggest that Topoisomerase II may control higher order chromatin architecture through direct binding interactions, independently of its well-known catalytic activity. J. Cell. Biochem. 69:127-142, 1998. © 1998 Wiley-Liss, Inc.
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  • 142
    ISSN: 0730-2312
    Keywords: haemochromatosis gene ; histone gene cluster ; YACs ; cosmid contig ; sequences ; species comparison ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The HFE (HLA-H) gene is a strong candidate gene for hereditary haemochromatosis and was localized on the short arm of chromosome 6 to 6p21.3-p22. In addition, the sequence of the homologous mouse and rat cDNA and a partial sequence from the mouse gene have been reported recently. In this report, we describe the location of the human and the mouse HFE (HLA-H) gene within the histone gene clusters on the human chromosome 6 and the mouse chromosome 13. Both the human and the murine gene were located on syntenic regions within the histone gene clusters in the vicinity of the histone H1t gene. The genomic sequence of the human HFE (HLA-H) gene and the 3′ portion of the homologous mouse gene were determined. Comparison of the genomic sequences from man and mouse and the cDNA sequence from rat shows significant similarities, also beyond the transcribed region of the mouse gene. J. Cell. Biochem. 69:117-126, 1998. © 1998 Wiley-Liss, Inc.
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  • 143
    ISSN: 0730-2312
    Keywords: HB-EGF ; cleavage-secretion ; PKC ; ErbB1 ; EGF receptor ; matrix metalloproteinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143-153, 1998. © 1998 Wiley-Liss, Inc.
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  • 144
    ISSN: 0730-2312
    Keywords: transcription ; mRNA stability ; dexamethasone ; gene regulation ; glucocorticoid receptor ; rat calvarial osteoblasts ; osteopontin ; vitamin D receptor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts. J. Cell. Biochem. 69:154-168, 1998. © 1998 Wiley-Liss, Inc.
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  • 145
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    Journal of Cellular Biochemistry 69 (1998), S. 169-180 
    ISSN: 0730-2312
    Keywords: growth factor ; bone ; osteoblast ; inflammation ; alkaline phosphatase ; differentiation ; proliferation ; PDGF ; mineralized nodules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Platelet-derived growth factor (PDGF) stimulates chemotaxis and proliferation of osteoblasts, and induces bone formation in vivo. To determine how PDGF might regulate these cells, the effect of PDGF on long-term mineralizing cultures of fetal rat osteoblastic cells was examined. Although PDGF increased cell proliferation in these cultures, continuous treatment with PDGF caused a dose-dependent decrease in mineralized nodule formation. When cells were treated with multiple, brief (1 day) exposures to PDGF at the osteoblast differentiation stage, there was a significant 50% increase in mineralized nodule area. Based on modulation of alkaline phosphatase activity it appears that longer-term exposure to PDGF reduces mineralized nodule formation largely by inhibiting differentiated osteoblast function, while short-term exposure enhances proliferation without inhibiting the differentiated phenotype. Thus, the ultimate affect of PDGF on bone formation is likely to reflect two processes: a positive effect through enhancing cell number or a negative effect by inhibiting differentiated function. The inhibitory effect of PDGF on formation of a mineralized matrix is unlikely to be simply a result of enhanced proliferation of “fibroblastic” cells since cultures treated with PDGF for 3 days and then transferred to new plastic dishes exhibited a 70% increase in mineralized nodule area compared to controls. These results would predict that multiple, brief exposures to PDGF would enhance bone formation in vivo, while prolonged exposure to PDGF, which is likely to occur in chronic inflammation, would inhibit differentiated osteoblast function and limit bone regeneration. J. Cell. Biochem. 69:169-180, 1998. © 1998 Wiley-Liss, Inc.
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  • 146
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    Journal of Cellular Biochemistry 69 (1998), S. 181-188 
    ISSN: 0730-2312
    Keywords: magnetic fields ; HSP70 gene expression ; human HSP70 promoter ; c-myc protein binding sites ; cellular stress ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated c-myc protein-binding sites on the HSP70 promoter as modulators of the induction of HSP70 gene expression in response to magnetic field stimulation (8μT at 60Hz) and whether the presence of c-myc protein potentiates transactivation of HSP70 expression. A 320 base pair region in the HSP70 promoter (+1 to -320) was analyzed. This region contains two c-myc-protein binding sites with consensus sequences located at -230 and -160 nucleotide positions (relative to the transcription initiation site) and overlapping with the region reported for the regulation of HSP70 gene expression by c-myc protein. This promoter region is upstream of other regulatory sequences, including the heat shock element (HSE), AP-2, and serum response element (SRE). Transfectants containing both c-myc protein-binding sites, HSP-MYC A and HSP-MYC B, and exposed to magnetic fields showed a 3.0-fold increase in expression of CAT activity as compared with sham-exposed control transfectants. Transfectants containing one c-myc binding site, HSP-MYC A, and exposed to magnetic fields showed a 2.3-fold increase in CAT expression. Transfectants in which both HSP-MYC A and HSP-MYC B binding sites were deleted showed no magnetic field sensitivity; values were virtually identical with sham-exposed controls. If the c-myc expression vector was not co-transfected with the constructs containing myc-binding sites, there was no difference in the expression of CAT activity between magnetically stimulated and sham-exposed controls, although both responded to heat shock. These data suggest that endogenous elevated levels of myc protein contribute to the induction of HSP70 in response to magnetic field stimulation. J. Cell. Biochem. 69:181-188, 1998. © 1998 Wiley-Liss, Inc.
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  • 147
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    Journal of Cellular Biochemistry 69 (1998), S. 189-200 
    ISSN: 0730-2312
    Keywords: peroxisome proliferator activated receptor ; retinoid x receptor ; retinoic acid receptor ; liver hyperplasia ; hepatocarcinoma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Chronic griseofulvin (GF) feeding induces preneoplastic foci followed by hepatocellular carcinoma in the mouse liver. Our previous study suggested that GF-induced hepatocellular proliferation had a different mechanism from that of peroxisome proliferator (PP)-induced direct hyperplasia. The GF-induced hepatocellular proliferation was mediated through activation of immediate early genes such as Fos, Jun, Myc, and NFκB. In contrast, PP-induced direct hyperplasia does not involve activation of any of these immediate early genes. It has been shown that nuclear hormone receptors including peroxisome proliferator activated receptors (PPARs) and retinoid x receptors (RXRs) play important roles in mediating the pleiotropic effects of PPs. To examine the possible roles of PPARs and RXRs during non-PP-induced hepatocellular proliferation and the interaction between PP and non-PP-induced proliferation, we have studied the expression of the PPAR and RXR genes in the GF model using northern blot hybridizations and gel retardation assays. The data showed that the expression of PPARα and RXRα genes was down-regulated in the livers containing preneoplastic nodules and in the liver tumors induced by GF. The mRNA down-regulation was accompanied by a decrease in the amount of nuclear protein-bound to peroxisome proliferator and retinoic acid responsive elements. Down-regulation was also associated with the suppressed expression of the PPARα/RXRα target genes (i.e., acyl-Co oxidase and cytochrome P450 4A1) and the catalase gene. The RXRγ gene was also down-regulated, but the RARα, β, and γ and PPARβ and γ genes were up-regulated. These results indicated that the hepatocarcinogenesis induced by GF is accompanied by suppression of the PPARα/RXRα-mediated direct hyperplasia pathway. The differential expression of these nuclear hormone receptors reveals a new aspect for understanding the individual roles and intercommunication of PPAR, RXR, and RAR isoforms in the liver. J. Cell. Biochem. 69:189-200, 1998. © 1998 Wiley-Liss, Inc.
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  • 148
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    Journal of Cellular Biochemistry 69 (1998), S. 201-210 
    ISSN: 0730-2312
    Keywords: sodium butyrate ; alkaline phosphatase ; A5 cells ; A5-DAP cells ; A5-BAG cells ; β-galactosidase ; retroviral vectors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have studied the effects of sodium butyrate (NaBu) on the expression of genes transduced by retroviral vectors and stably expressed in two salivary gland-derived cell lines, A5-DAP and A5-BAG, established earlier. These cell lines were obtained by infecting A5 cells with the retroviral vectors DAP and BAG, respectively, and by selecting neomycin-resistant transduced cells. A5-DAP cells express human placental alkaline phosphatase (PLAP) and A5-BAG cells bacterial β-galactosidase, both under the control of the viral long terminal repeat (LTR) enhancer-promoter. NaBu in the concentration of 2-8 mM inhibited the growth of A5-DAP cells, and induced the expression of heat-stable PLAP. These effects of NaBu were dose-dependent. Induction of PLAP in clones of A5-DAP cells that express different basal levels of the enzyme was not correlated with the relative inducibilty by NaBu. Exposure to 4 mM NaBu for 48 h increased the PLAP mRNA level by 31%. A5-DAP cells released, in a time-dependent manner, PLAP into the culture medium. Cells treated with NaBu released more PLAP than untreated cells in proportion to their elevated level of the enzyme. The parent A5 cells also express a low level of tissue non-specific type alkaline phosphatase, which was also induced by NaBu. NaBu inhibited the growth of A5-BAG cells also, and increased the β-galactosidase level. These data indicate the genes transduced by retroviral vectors can be induced by NaBu, which most likely interacts with the viral LTR. J. Cell. Biochem. 69:201-210, 1998. © 1998 Wiley-Liss, Inc.
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  • 149
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    Journal of Cellular Biochemistry 69 (1998), S. 211-220 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; protein kinase CK2 ; disulfide bonds ; sodium tetrathionate ; iodoacetamide ; sulfhydryl crosslinking ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nuclear matrix (NM) appears to be an intranuclear locale for significant and dynamic association of the ubiquitous multifunctional messenger-independent serine/threonine protein kinase CK2 that has been implicated in growth control [Tawfic et al. (1996): J Cell Biochem 61:165-171]. We have examined the nature of the association of CK2 with the NM. Nuclei prepared in the presence of a sulfhydryl-blocking reagent such as iodoacetamide demonstrate a reduction in the amount of CK2 associated with the NM to less than 5% of the control. On the other hand, when nuclei are treated with the sulfhydryl crosslinking reagent sodium tetrathionate, NM-associated CK2 increases severalfold. Treatment of nuclei with sodium tetrathionate followed by 2-mercaptoethanol blocks this increase. Nuclei isolated from rat liver and prostate behaved similarly, suggesting an identical mode of association of CK2 with the NM regardless of the organ. These results indicate a role of sulfhydryl interactions such that NM anchoring of CK2 occurs via its β subunit, which contains several vicinal cysteine residues. Further, various sulfhydryl-blocking reagents inhibited CK2 activity in a concentration-dependent manner, and the inhibitory effect was reversed by agents such as dithiothreitol, implying that cysteine residues in the CK2 play a role in its catalytic activity. J. Cell. Biochem. 69:211-220, 1998. Published 1998 Wiley-Liss, Inc.
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  • 150
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    Journal of Cellular Biochemistry 69 (1998), S. 221-231 
    ISSN: 0730-2312
    Keywords: sodium fluoride ; stress response ; stress proteins ; heat shock proteins ; rat brain tumor 9L cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221-231, 1998. © 1998 Wiley-Liss, Inc.
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  • 151
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    Journal of Cellular Biochemistry 69 (1998), S. 233-243 
    ISSN: 0730-2312
    Keywords: histamine ; polyamines ; cytochrome P450 ; cell growth ; cell proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Histamine and polyamines have been implicated in the mediation of cell proliferation. Our previous work linked the growth-modulatory effects of histamine with its binding to intracellular sites in microsomes and nuclei of various tissues. In this study, we identify cytochrome P450 enzymes as a major component of microsomal intracellular sites in hepatocytes and demonstrate that polyamines compete with high affinity for histamine binding to them. Spectral measurement of histamine binding to P450 in liver microsomes resolved high and intermediate affinity binding sites (Ks1 = 2.4 ± 1.6 μM; Ks2 = 90 ± 17 μM) that corresponded to microsomal binding sites (Kd1 = 1.0 ± 0.9 μM; Kd2 = 57 ± 13 μM) resolved by 3H-histamine binding; additional low affinity (Kd3 ∼ 3 mM), and probably physiologically irrelevant, sites were resolved only by 3H-histamine radioligand studies. As determined spectrally, treatment of microsomes with NADPH/carbon monoxide decreased histamine binding to P450 by about 90% and, as determined by 3H-histamine binding, abolished the high affinity sites and reduced by 85% the number of intermediate sites. Spermine competed potently for 3H-histamine binding: in microsomes, Ki = 9.8 ± 5.8 μM; in nuclei, Ki = 13.7 ± 3.1 μM; in chromatin, Ki = 46 ± 33 nM. Polyamines inhibited the P450/histamine absorbance complex with the rank order of potency: spermine 〉 spermidine ≫ putrescine. In contrast, histamine did not compete for 3H- spermidine binding in nuclei or microsomes, suggesting that polyamines modulate histamine binding allosterically. We propose that certain P450 isozymes that modulate gene function by controlling the level of oxygenated lipids, represent at least one common intracellular target of growth-regulatory endogenous bioamines and, as shown previously, of exogenous growth-modulatory drugs including antiestrogens, antiandrogens, and certain antidepressants and antihistamines. J. Cell. Biochem. 69:233-243, 1998. © 1998 Wiley-Liss, Inc.
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  • 152
    ISSN: 0730-2312
    Keywords: monomeric laminin receptor ; receptor maturation ; acylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule. J. Cell. Biochem. 69:244-251, 1998. © 1998 Wiley-Liss, Inc.
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  • 153
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    Journal of Cellular Biochemistry 69 (1998), S. 252-259 
    ISSN: 0730-2312
    Keywords: bisphosphonate ; prostaglandin F2α ; interleukin-6 ; phospholipase D ; osteoblast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In previous studies, we have reported that PGF2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein in osteoblast-like MC3T3-E1 cells, and that PGF2α and PGE1 induce interleukin-6 (IL-6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF2α- and PGE1-induced IL-6 synthesis in these cells. Tiludronate significantly suppressed the PGF2α-induced IL-6 secretion in a dose-dependent manner in the range between 0.1 and 30 μM. However, the IL-6 secretion induced by PGE1 or (Bu)2cAMP was hardly affected by tiludronate. The choline formation induced by PGF2α was reduced by tiludronate dose-dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF2α-induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP-binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF-induced IL-6 secretion in human osteoblastic osteosarcoma Saos-2 cells. These results strongly suggest that tiludronate inhibits PGF2α-induced IL-6 synthesis via suppression of phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP-binding protein and phospholipase D. J. Cell. Biochem. 69:252-259, 1998. © 1998 Wiley-Liss, Inc.
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  • 154
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    Journal of Cellular Biochemistry 69 (1998), S. 260-270 
    ISSN: 0730-2312
    Keywords: oncogenic function of mutant p53 ; MAR-DNA elements ; MAR-DNA binding by mutant p53 ; MethA p53 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We recently reported that murine MethA mutant but not wild-type p53 specifically binds to MAR-DNA elements (MARs) with high affinity. Here we show that this DNA binding activity is exerted not only by MethA mutant p53 but also by other murine mutant p53 proteins isolated from the transformed murine BALB/c cell lines 3T3tx and T3T3 and differing in their conformational status. High affinity MAR-DNA binding was not restricted to the XbaI-IgE-MAR-DNA fragment from the murine immunoglobulin heavy chain gene enhancer locus [Cockerill et al. (1987): J Biol Chem 262:5394-5397] used in previous studies, as MethA p53 also specifically interacted with other A/T-rich bona fide MARs. Not only murine but also human mutant p53 proteins carrying the mutational hot spot amino acid exchanges 175Arg→His, 273Arg→Pro, or 273Arg→His bound to the XbaI-IgE-MAR-DNA fragment. We therefore conclude that high affinity MAR-DNA binding is a property common to a variety of mutant p53 proteins. J. Cell. Biochem. 69:260-270, 1998. © 1998 Wiley-Liss, Inc.
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  • 155
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    Journal of Cellular Biochemistry 69 (1998), S. 282-290 
    ISSN: 0730-2312
    Keywords: estrogen modulation ; osteoblastic cells ; plasma membrane receptors ; nuclear receptors ; gap junction communication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Two osteoblastic cell populations, calvarial and marrow stromal cells, were exposed to estrogen derivatives in vitro. The hormonal effect was monitored by following intracellular Ca+2 levels [Ca+2]i and gap-junction communication. We measured fast changes in intracellular Ca+2 levels in response, of these cells, to the steroid hormones. The changes were dose dependent revealing maximal activity at 100 pM by 17-β-Estradiol and 1 nM by estradiol-CMO. Additionally, the effect of estrogen, on functional coupling of the cells, was measured using fluorescence dye migration and counting the number of neighboring cells coupled by gap junctions. An uncoupling effect was demonstrated in response of these cells to estrogen treatment. The quick stereospecific effect was achieved in the presence of 17-β-estradiol but not in the presence of 17-α-estradiol. These results suggest the involvement of plasma membrane receptors in addition to the already known nuclear receptors in transducing the hormone effects in the osteoblastic cells. J. Cell. Biochem. 69:282-290, 1998. © 1998 Wiley-Liss, Inc.
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  • 156
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    Journal of Cellular Biochemistry 69 (1998), S. 291-303 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; TGF-β1 ; bone ; osteoblast differentiation ; mineralization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4-6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β-treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β-treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β-dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291-303, 1998. © 1998 Wiley-Liss, Inc.
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  • 157
    ISSN: 0730-2312
    Keywords: butyrate ; isobutyramide ; prostate cancer ; LNCaP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Progression to androgen independence remains the main obstacle to improving survival and quality of life in patients with advanced prostate cancer. Induction of differentiation may serve as a rational basis for prevention of progression to androgen independence by modulating gene expression activated by castration or upregulated during androgen-independent progression. The objectives of this study were to characterize the in vitro effects of sodium butyrate on human prostate cancer cell growth, PSA gene expression, and differentiation in the LNCaP tumor model and to determine whether tumor progression in vivo is delayed by isobutyramide, an orally bioavailable butyrate analogue with a longer half-life. The effects of isobutyramide on LNCaP tumor growth and serum PSA levels in both intact and castrate male mice were compared to controls. At concentrations 〉 1 mM, butyrate induced dose-dependent changes towards a more differentiated phenotype, G1 cell cycle arrest, and an 80% decrease in LNCaP cell growth rates. PSA gene expression was increased threefold by butyrate, indicative of differentiation-enhanced gene expression. The half-life of isobutyramide in athymic mice was determined by gas chromatography to be 4 h. During a 4 week period in intact-placebo mice, tumor volume and serum PSA increased 4.1- and 6.6-fold, respectively, compared to twofold and 2.7-fold increases in tumor volume and serum PSA in intact-treated mice. During a 7 week period in castrate-placebo mice, tumor volume and serum PSA levels increased 2.4-fold and fourfold, respectively, compared to a 50% reduction in tumor volume and a twofold increase in serum PSA above nadir levels in castrate mice treated with adjuvant isobutyramide. Isobutyramide treatment induced pronouced morphological changes in LNCaP tumor cells, with loss of defined nucleoli and dispersion of chromatin distribution. LNCaP tumor PSA mRNA levels actually increased threefold, indicative of differentiation-enhanced gene expression. This study demonstrates that butyrate causes LNCaP cell cycle arrest and increased PSA gene expression, both indicative of differentiation. The combination of castration and adjuvant isobutyramide was synergistic in delaying tumor progression. Decreased tumor cell proliferation and increased PSA gene expression induced by isobutyramide results in disconcordant changes in serum PSA and tumor volume and reduces the utility of serum PSA as a marker of response to therapy. J. Cell. Biochem. 69:271-281, 1998. © 1998 Wiley-Liss, Inc.
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  • 158
    ISSN: 0730-2312
    Keywords: VAT-1 ; Pacific electric ray Torpedo californica ; ATPase ; Mus musculus ; gene structure ; Ehrlich ascites tumor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recently, interest has focused on the human gene encoding the putative protein homologous to VAT-1, the major protein of the synaptic vesicles of the electric organ of the Pacific electric ray Torpedo californica, after it has been localized on chromosome locus 17q21 in a region encompassing the breast cancer gene BRCA1. Chromosomal instability in this region is implicated in inherited predisposition for breast and ovarian cancer. Here we describe isolation and biochemical characterization of a mammalian 48 kDa protein homologous to the VAT-1 protein of Torpedo californica. This VAT-1 homolog was isolated from a murine breast cancer cell line (Ehrlich ascites tumor) and identified by sequencing of cleavage peptides. The isolated VAT-1 homolog protein displays an ATPase activity and exists in two isoforms with isoelectric points of 5.7 and 5.8. cDNA was prepared from Ehrlich ascites tumor cells, and the murine VAT-1 homolog sequence was amplified by polymerase chain reaction and partially sequenced. The known part of the murine and the human translated sequences share 97% identity. By Northern blots, the size of the VAT-1 homolog mRNA in both murine and human (T47D) breast cancer cells was determined to be 2.8 kb. Based on the presented data, a modified gene structure of the human VAT-1 homolog with an extended exon 1 is proposed. VAT-1 and the mammalian VAT-1 homolog form a subgroup within the protein superfamily of medium-chain dehydrogenases/reductases. J. Cell. Biochem. 69:304-315, 1998. © 1998 Wiley-Liss, Inc.
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  • 159
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    Journal of Cellular Biochemistry 69 (1998), S. 326-335 
    ISSN: 0730-2312
    Keywords: copper ; human endothelial cells ; angiogenesis ; growth factors ; cell proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 μM CuSO4 in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-α, transforming growth factor-β, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1α. Moreover, despite the previous observations that copper increased total specific binding of 125I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis. J. Cell. Biochem. 69:326-335, 1998. © 1998 Wiley-Liss, Inc.
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  • 160
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    Journal of Cellular Biochemistry 69 (1998), S. 316-325 
    ISSN: 0730-2312
    Keywords: osteoprogenitors ; mineralization ; marrow stroma ; Src ; tyrosine kinase dexamethasone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Src protein is essential for the regulation of bone turnover primarily via bone resorption because it is required in osteoclast differentiation and function. We followed temporal changes of Src protein abundance in marrow stromal cells induced to mineralize by dexamethasone (DEX), growth in cold temperature, or both. Given the tyrosine kinase function of Src and its numerous substrates, profiles of phosphotyrosine-containing proteins were followed as well. On day 11 of stimulation, specific alkaline phosphatase (ALP) activity at 30°C decreased under DEX relative to 37°C cultures, in accord with increased cell counts. Mineralization per well under DEX increased by 25% at 37°C, whereas at 30°C it increased by more than threefold regardless of the DEX stimulation. At 30°C, on a per cell basis mineralization increased 2.5 and 3 times with and without DEX, respectively. Cultures at 37°C showed a general drop per cell of many phosphotyrosine-containing proteins on day 3 relative to days 1 and 2 in both DEX-stimulated and nonstimulated cultures; several proteins did recover (recuperate) thereafter. On days 1 and 2, the phosphotyrosine signal was higher in several proteins under DEX stimulation; this trend became inverted after day 3. The changes in abundance per cell of Src protein (pp60src) followed a similar trend, and in addition a truncated Src molecule, p54/52src, was detected as a putative cleavage product presumably representing its carboxy terminus. The pp60src was most abundant, relative to its truncated product, in day 7 nonstimulated cultures, whereas under DEX stimulation the truncated species pp54/52src showed the highest relative abundance on days 7. At 30°C, DEX stimulation accentuated the increase in Src protein on day 3, showed no change on day 7, and returned to increase Src protein on day 10. Potassium ionophorvalinomycin, considered to select against mineralizing osteoprogenitors at 30°C, showed on day 10 in the absence of DEX a relative increase in truncated Src protein compared to both DEX-stimulated and nonstimulated cultures in the absence of valinomycin. On day 7 of DEX stimulation, the presence of valinomycin resulted in low p54/52src. Among phosphotyrosine-containing proteins, a 32-34 kDa band, as yet unidentified, showed the most concordant changes with mineralization induction. P32-34 decreased by DEX on days 2 and 8 and increased by low temperature alone or combined with DEX on day 3. On day 7, p32-34 did not change under DEX, but valinomycin selected cells with less phoshpotyrosine-containing p32-34. Taken together, high Src abundance at the start of osteogenic induction followed by a decrease 1 week later is probably related to energy metabolism-dependent induction of mineralization. This is in temporal accord with the increase in Src truncation and fluctuation in mitochondrial membrane potential (which affects mineralization). The reported binding of amino-terminal Src oligopeptide to p32 ADP/ATP carrier in the mitochondrial inner membrane raises the question of its possible involvement in mitochondria-regulated mineralization. J. Cell. Biochem. 69:316-325, 1998. © 1998 Wiley-Liss, Inc.
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  • 161
    ISSN: 0730-2312
    Keywords: architectural transcription factor ; nuclear matrix ; osteoblast ; parathyroid hormone ; type I collagen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In connective tissue, cell structure contributes to type I collagen expression. Differences in osteoblast microarchitecture may account for the two distinct cis elements regulating basal expression, in vivo and in vitro, of the rat type I collagen α1(I) polypeptide chain (COL1A1). The COL1A1 promoter conformation may be the penultimate culmination of osteoblast structure. Architectural transcription factors bind to the minor groove of AT-rich DNA and bend it, altering interactions between other trans-acting proteins. Similarly, nuclear matrix (NM) proteins bind to the minor groove of AT-rich matrix-attachment regions, regulating transcription by altering DNA structure. We propose that osteoblast NM architectural transcription factors link cell structure to promoter geometry and COL1A1 transcription. Our objective was to identify potential osteoblast NM architectural transcription factors near the in vitro and in vivo regulatory regions of the rat COL1A1 promoter. Nuclear protein-promoter interactions were analyzed by gel shift analysis and related techniques. NM extracts were derived from rat osteosarcoma cells and from rat bone. The NM protein, NMP4, and a soluble nuclear protein, NP, both bound to two homologous poly(dT) elements within the COL1A1 in vitro regulatory region and proximal to the in vivo regulatory element. These proteins bound within the minor groove and bent the DNA. Parathyroid hormone increased NP/NMP4 binding to both poly(dT) elements and decreased COL1A1 mRNA in the osteosarcoma cells. NP/NMP4-COL1A1 promoter interactions may represent a molecular pathway by which osteoblast structure is coupled to COL1A1 expression. J. Cell. Biochem. 69:336-352. © 1998 Wiley-Liss, Inc.
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  • 162
    ISSN: 0730-2312
    Keywords: AML/CBF/PEBP2 ; CBFa1 ; differentiation ; osteoblasts ; regulatory elements ; transforming growth factor-β ; receptor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Organization of the transforming growth factor-β (TGF-β) type I receptor (TRI) promoter predicts constitutive transcription, although its activity increases with differentiation status in cultured osteoblasts. Several sequences in the rat TRI promoter comprise cis-acting elements for CBFa (AML/PEBP2α) transcription factors. By gel mobility shift and immunological analyses, a principal osteoblast-derived nuclear factor that binds to these sites is CBFa1(AML-3/PEBP2αA). Rat CBFa1 levels parallel expression of the osteoblast phenotype and increase under conditions that promote mineralized bone nodule formation in vitro. Fusion of CBFa binding sequence from the TRI promoter to enhancer-free transfection vector increases reporter gene expression in cells that possess abundant CBFa1, and overexpression of CBFa increase the activity of transfected native TRI promoter/reporter plasmid. Consequently, phenotype-restricted use of cis-acting elements for CBFa transcription factors can contribute to the high levels of TRI that parallel osteoblast differentiation and to the potent effects of TGF-β on osteoblast function. J. Cell. Biochem. 69:353-363. © 1998 Wiley-Liss, Inc.
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  • 163
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    Journal of Cellular Biochemistry 69 (1998), S. 364-375 
    ISSN: 0730-2312
    Keywords: IGF ; IGFBP ; zinc ; IGFBP-3 ; IGFBP-5 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of multivalent cations on [125I]-IGF binding to cell-associated IGFBPs was investigated using human fibroblasts. The major cell-associated binding site for [125I]-IGF-I is IGFBP-3 and for [125I]-IGF-II are IGFBP-3 and IGFBP-5. Lanthanum and chromium did not affect either [125I]-IGF-I or [125I]-IGF-II binding to cell-associated IGFBPs. By contrast, zinc (Zn2+), gold (Au3+), and cadmium (Cd2+) depressed binding of both ligands. Ligand binding resulted in nonlinear Scatchard plots. Assuming a pre-existent asymmetric model with high- (KaHi) and low- (KaLo) affinity sites, Zn2+ lowered both KaHi and KaLo. Au3+ eliminated KaHi. Assuming that the nonlinear plots were caused by ligand-induced negative cooperativity, Zn2+ and Cd2+ lowered both Ke and Kf (affinity of unoccupied and saturated IGFBPs, respectively). Au3+ eliminated Ke and reduced Kf. Zn2+ was active at serum levels in lowering IGF binding. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) indicating similar mechanisms of action. A zinc-binding motif is present in the IGFBPs, but not in the IGFs. We demonstrate for the first time that the trace nutrient zinc and related multivalent cations decrease IGF binding to fibroblast-associated IGFBPs by lowering the affinity of the IGF-IGFBP interaction. J. Cell. Biochem. 69:364-375, 1998. © 1998 Wiley-Liss, Inc.
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  • 164
    ISSN: 0730-2312
    Keywords: homeobox ; mammary gland ; morphogenesis ; basement membrane ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Homeobox-containing genes encode transcriptional regulators involved in cell fate and pattern formation during embryogenesis. Recently, it has become clear that their expression in continuously developing adult tissues, as well as in tumorigenesis, may be of equal importance. In the mouse mammary gland, expression patterns of several homeobox genes suggest a role in epithelial-stromal interactions. Because the stroma and the extracellular matrix (ECM) are known to influence both functional and morphological development of the mammary gland, we asked whether these genes would be expressed postnatally in the gland and also in cell lines in culture and whether they could be modulated by ECM. Using a polymerase chain reaction-base strategy five members of the Hox gene clusters a and b were shown to be expressed in cultured mouse mammary cells. Hoxa-1 and Hoxb-7 were chosen for further analysis. Hoxb-7 was chosen because it had not been described previously in the mammary gland and was modulated at different stages of gland development. Hoxa-1 was chosen because it was reported previously to be expressed only in mammary tumors, and not in normal glands. We showed that culturing the mammary epithelial cell lines SCp2 and CID-9 on a basement membrane (BM) that was previously shown to induce a lactational phenotype was necessary to turn off Hoxb-7, but a change in cell shape, brought about by culturing the cells on an inert substratum such as polyHEMA, was sufficient to downregulate Hoxa-1. This is the first report of modulation of homeobox genes by ECM. The results provide a rationale for the differential pattern of expression in vivo of Hoxa-1 and Hoxb-7 during different stages of development. The culture model should permit further in-depth analysis of the molecular mechanisms involved in how ECM signaling and homeobox genes may interact to bring about tissue organization. J. Cell. Biochem. 69:377-391, 1998. © 1998 Wiley-Liss, Inc.
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  • 165
    ISSN: 0730-2312
    Keywords: chondrocytes ; cyclooxygenase-2 ; c-Jun N-terminal kinase ; protein kinase A ; cAMP response element ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The involvement of serine/threonine protein phosphatases in signaling pathways that control the expression of the cyclooxygenase-2 (COX-2) gene in human chondrocytes was examined. Okadaic acid (OKA), an inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A), induced a delayed, time-dependent increase in the rate of COX-2 gene transcription (runoff assay) resulting in increased steady-state mRNA levels and enzyme synthesis. The latter response was dose dependent over a narrow range of 1-30 nmol/L with declining expression and synthesis of COX-2 at higher concentrations due to cell toxicity. The delayed increase in COX-2 mRNA expression was accompanied by the induction of the proto-oncogenes c-jun, junB, junD, and c-fos (but not FosB or Fra-1). Increased phosphorylation of CREB-1/ATF-1 transcription factors was observed beginning at 4 h and reached a zenith at 8 h. Gel-shift analysis confirmed the up-regulation of AP-1 and CRE nuclear binding proteins, though there was little or no OKA-induced nuclear protein binding to SP-1, AP-2, NF-κB or NF-IL-6 regulatory elements. OKA-induced nuclear protein binding to 32P-CRE oligonucleotides was abrogated by a pharmacological inhibitor of protein kinase A (PKA), KT-5720; the latter compound also inhibited OKA-induced COX-2 enzyme synthesis. Calphostin C (CalC), an inhibitor of PKC isoenzymes, had little effect in this regard. Inhibition of 32P-CRE binding was also observed in the presence of an antibody to CREB-binding protein (265-kDa CBP), an integrator and coactivator of cAMP-responsive genes. The binding to 32P-CRE was unaffected in the presence of excess radioinert AP-1 and COX-2 NF-IL-6 oligonucleotides, although a COX-2 CRE-oligo competed very efficiently. 32P-AP-1 consensus sequence binding was unaffected by incubation of chondrocytes with KT-5720 or CalC, but was dramatically diminished by excess radioinert AP-1 and CRE-COX-2 oligos. Supershift analysis in the presence of antibodies to c-Jun, c-Fos, JunD, and JunB suggested that AP-1 complexes were composed of c-Fos, JunB, and possibly c-Jun. OKA has no effect on total cellular PKC activity but caused a delayed time-dependent increase in total PKA activity and synthesis. OKA suppressed the activity of the MAP kinases, ERK1/2 in a time-dependent fashion, suggesting that the Raf-1/MEKK1/MEK1/ERK1,2 cascade was compromised by OKA treatment. By contrast, OKA caused a dramatic increase in SAPK/JNK expression and activity, indicative of an activation of MEKK1/JNKK/SAPK/JNK pathway. OKA stimulated a dose-dependent activation of CAT activity using transfected promoter-CAT constructs harboring the regulatory elements AP-1 (c-jun promoter) and CRE (CRE-tkCAT). We conclude that in primary phenotypically stable human chondrocytes, COX-2 gene expression may be controlled by critical phosphatases that interact with phosphorylation dependent (e.g., MAP kinases:AP-1, PKA:CREB/ATF) signaling pathways. AP-1 and CREB/ATF families of transcription factors may be important substrates for PP-1/PP-2A in human chondrocytes. J. Cell. Biochem. 69:392-413, 1998. © 1998 Wiley-Liss, Inc.
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  • 166
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    Journal of Cellular Biochemistry 69 (1998), S. 425-435 
    ISSN: 0730-2312
    Keywords: perforin ; cell cycle ; apoptosis ; T lymphocyte ; DNA synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G1/S interface. Cells were arrested at the G1/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G1/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G1 to G2cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G1 phase of the cell cycle are less susceptible to the perforin pathway than cells in G2and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425-435, 1998. © 1998 Wiley-Liss, Inc.
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  • 167
    ISSN: 0730-2312
    Keywords: YKL40 ; purification ; guinea pig ; chondrocytes ; biochemical characterization ; regulation ; insulin-like growth factors ; osteoarthritis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The aim of this study was to purify, characterize, and study the regulation at the chondrocyte level of the guinea pig (gp) homologue of human (R) YKL40, a putative marker of arthritic disorders. Studying YKL40 in guinea pigs is of particular interest, as age-related osteoarthritis develops in this species spontaneously. Both N-terminal sequencing and total amino acid composition of gpYKL40 purified from the secretion medium of cultured articular chondrocytes indicate a high degree of identity with hYKL40. gpYKL40 was found to contain complex N-linked carbohydrate, as demonstrated by N-glycosidase F and endoglycosidase F digestion. Isoelectric focusing demonstrated the presence of a major band at pI 6.7. The secretion of gpYKL40 by confluent articular chondrocytes in the extracellular medium was studied by immunoblotting. gpYKL40 was released by chondrocytes continuously over a 7 day period and did not appear to be degraded by proteinases, as its signal intensity in cell-free medium at 37°C did not decrease with time. Thus, gpYKL40 displays high stability and accumulates in extracellular medium without reaching a steady-state level. Among the main factors known to regulate cartilage metabolism, IL-1β, TNF-α, bFGF, or 1,25(OH)2D3 did not alter the basal level of gpYKL40, and retinoic acid had a slight inhibitory effect; TGF-β and IGF-I and -II dose-dependently and inversely modulated this basal level. TGF-β at 5 ng/ml decreased extracellular gpYKL40 2.9-fold, whereas IGF-I and IGF-II at 50 ng/ml increased extracellular gpYKL40 3.6- and 3.4-fold, respectively. The present biochemical and biological findings give new insights for studying the function of YKL40 in cartilage. J. Cell Biochem. 69:414-424, 1998. © 1998 Wiley-Liss, Inc.
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  • 168
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    Keywords: small heat shock proteins ; TNFα ; phosphorylation mutant ; SB203580 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The role of murine Hsp25 phosphorylation in the protection mediated by this protein against TNFα- or H2O2-mediated cytotoxicity was investigated in L929 cell lines expressing wild type (wt-) or nonphosphorylatable (mt-) Hsp25. We show that mt-Hsp25, in which the phosphorylation sites, serines 15 and 86, were replaced by alanines, is still efficient in decreasing intracellular reactive oxygen species levels and in raising glutathione cellular content, leading the protective activity of mt-Hsp25 against oxidative stress to be identical to that of wt-Hsp25. To independently investigate the role of Hsp25 phosphorylation, we blocked TNFα-induced phosphorylation of wt-Hsp25 using SB203580, a specific inhibitor of the P38 MAP kinase. This treatment did not abolish the protective activity of Hsp25 against TNFα. The pattern of Hsp25 oligomerization was also analyzed, showing mt-Hsp25 to constitutively display large native sizes, as does wt-Hsp25 after TNFα treatment in the presence of SB203580. Our results, therefore, are consistent with the possibility that the hyperaggregated form of Hsp25 is responsible for the protective activity against oxidative stress and that the phosphorylation of serines 15 and/or 86 by interfering with this structural reorganization, may lead to the inactivation of Hsp25 protective activity. J. Cell. Biochem. 69:436-452, 1998. © 1998 Wiley-Liss, Inc.
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  • 169
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    Keywords: Rous sarcoma virus ; chondrocytes ; matrix calcification ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification. J. Cell. Biochem. 69:453-462, 1998. © 1998 Wiley-Liss, Inc.
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  • 170
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    Journal of Cellular Biochemistry 69 (1998), S. 463-469 
    ISSN: 0730-2312
    Keywords: Adriamycin ; rat hepatoma ; ρ° cells ; multidrug resistance ; P-glycoprotein ; Sandoz SDZ PSC 833 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Rat hepatoma cells lacking mitochondrial DNA (ρ° cells) were used as a model system to examine the possible roles of mitochondrial DNA as a target for the DNA-acting anticancer drug Adriamycin (doxorubicin). The ρ° cells were 45-fold less sensitive to Adriamycin than the parental ρ+ cells containing mitochondrial DNA. Other non-DNA-acting drugs also exhibited similar behaviour, and this was shown to be due to a multidrug resistance (MDR) phenotype in the ρ° cells. This was indicated by confocal microscopy where ρ+ cells exhibited thirteenfold higher cellular levels of Adriamycin than ρ° cells. Upregulation (tenfold) of P-glycoprotein in ρ° cells was also confirmed by Northern dot blot analysis. Since the MDR phenotype is present in ρ° cells and upregulation of P-glycoprotein is maintained in these cells, ρ° cells are not a good model system for drug-DNA studies (where the drug is susceptible to extrusion by P-glycoprotein), and any such results obtained with this system must be treated with considerable caution. J. Cell. Biochem. 69:463-469, 1998. © 1998 Wiley-Liss, Inc.
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  • 171
    ISSN: 0730-2312
    Keywords: Cordyceps sinensis ; adrenal cells ; steroidogenesis ; signal pathway ; PKC ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cordyceps sinensiscontains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus-pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water-soluble extract of Cordyceps sinensis(CS) and thesignaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose-dependent manner by CS, reaching a maximun at 25 μg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS-induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS-treated cells. However, combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS-induced steroidogenesis. J. Cell. Biochem. 69:483-489, 1998. © 1998 Wiley-Liss, Inc.
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  • 172
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    Journal of Cellular Biochemistry 69 (1998), S. 470-482 
    ISSN: 0730-2312
    Keywords: enterocytes ; 1,25(OH)2-vitamin D3 ; tyrosine phosphorylation ; MAP kinase activation ; VDRnuc ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The steroid hormone 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1α,25(OH)2D3 action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1α,25(OH)2D3 demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42-44, 55-60, and 105-120 Kda. The 42-44 Kda bands comigrated with mitogen-activated protein (MAP) kinase (immunoblotting with anti-MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose-dependent (0.01-10 nM), with maximal stimulation at 1 nM (three- to fivefold). This effect was specific for 1α,25(OH)2D3 since its metabolic precursors 25(OH)D3and vitamin D3 did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1α,25(OH)2D3-induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormone's effects by 30%. We have evaluated the ability of 1α,25(OH)2D3 analogs, which have complete flexibility around the 6,7 carbon-carbon bond (6F) or which are locked in either the 6-s-cis (6C) or the 6-s-trans(6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1β,25(OH)2D3, a known antagonist of 1α,25(OH)2D3-mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1α,25(OH)2D3 and analogs which can achieve the 6-s-cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes. J. Cell. Biochem. 69:470-482, 1998. © 1998 Wiley-Liss, Inc.
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  • 173
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    Journal of Cellular Biochemistry 69 (1998), S. 490-505 
    ISSN: 0730-2312
    Keywords: nucleolus ; nuclear import ; ribosomal protein L5 ; ribonucleoprotein particles ; ribosome assembly ; TFIIIA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, 32P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, 32P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of 35S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule. J. Cell. Biochem. 69:490-505, 1998. © 1998 Wiley-Liss, Inc.
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  • 174
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    Journal of Cellular Biochemistry 69 (1998), S. 506-521 
    ISSN: 0730-2312
    Keywords: heart ; development ; CaMPK ; cAPK ; CDK ; cGPK ; Kkialre ; PKC ; Wee1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: During early postnatal development, cardiomyocytes, which comprise about 80% of ventricular mass and volume, become phenotypically developed to facilitate their contractile functions and terminally differentiated to grow only in size but not in cell number. These changes are due to the expression of contractile proteins as well as the regulation of intracellular signal transduction proteins. In this study, the expression patterns of several protein kinases involved in various cardiac functions and cell-cycle control were analyzed by Western blotting of ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats. The expression level of cAMP-dependent protein kinase was slightly decreased (20%) over the first year, whereas no change was detected in cGMP-dependent protein kinase I. Calmodulin-dependent protein kinase II, which is involved in Ca2+ uptake into the sarcoplasmic reticulum, was increased as much as ten-fold. To the contrary, the expressions of protein kinase C-α and ι declined 77% with age. Cyclin-dependent protein kinases (CDKs) such as CDK1, CDK2, CDK4, and CDK5, which are required for cell-cycle progression, abruptly declined to almost undetectable levels after 10-20 days of age. In contrast, other CDK-related kinases, such as CDK8 or Kkialre, did not change significantly or increased up to 50% with age, respectively. Protein kinases implicated in CDK regulation such as CDK7 and Wee1 were either slightly increased in expression or did not change significantly. All of the proteins that were detected in ventricular extracts were also identified in isolated cardiac myocytes in equivalent amounts and analyzed for their relative expression in ten other adult rat tissues. J. Cell. Biochem. 69:506-521, 1998. © 1998 Wiley-Liss, Inc.
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  • 175
    ISSN: 0730-2312
    Keywords: apoptosis ; p53 ; pRb2/p130 ; E2F ; transcriptional control ; leukemia ; protein phosphatases ; colon cancer ; retinoblastoma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A significant portion of published literature is dedicated to describing the cloning and the characterization of proteins involved in the progression of the cell cycle, which govern cell growth both in cancer and normal ontogenesis. With this abundance of information, the cascading pathways of molecular events that occur in the cell cycle are proving to be exceedingly complicated. The purpose of this conference was to attract the leading clinical and basic science investigators in the growth control field with a final goal to determine how this current wealth of knowledge can be used to impact upon patient care and management by the design of novel adjuvant therapeutics specifically targeted at tumor cells and the identification of molecular diagnostic and/or prognostic markers in an efficient and cost effective manner. J. Cell. Biochem. 70:1-7, 1998. © 1998 Wiley-Liss, Inc.
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  • 176
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    Journal of Cellular Biochemistry 70 (1998), S. 8-21 
    ISSN: 0730-2312
    Keywords: activin A ; bone marrow stromal cells ; gene regulation ; promoter activity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Activin A, a member of the TGF-β superfamily, plays roles in differentiation and development, including hematopoiesis. Our previous studies indicated that the expression of activin A by human bone marrow cells and monocytes is highly regulated by inflammatory cytokines and glucocorticoids. The present study was undertaken to investigate the regulation of activin A gene expression in the human bone marrow stromal cell lines L87/4 and HS-5, as well as in primary stromal cells. Northern blots demonstrated that, like primary stromal cells, the cell lines expressed four activin A RNA transcripts (6.4, 4.0, 2.8, and 1.6 kb), although distribution of the RNA among the four sizes varied. The locations of the 5′ ends of the RNAs were investigated by Northern blots and RNase protection assays. The results identified a transcription start site at 212 nucleotides upstream of the translation start codon. In addition, luciferase expression assays of a series of deletion constructs were used to identify regulatory sequences upstream of the activin A gene. A 58 bp upstream sequence exhibits promoter activity. However, severalfold higher expression requires a positive element consisting of an additional 71 bp of the upstream region. Promoter activity was also identified between 2.5 and 3.6 kb upstream of the start codon. These findings suggest that expression of activin A at the transcriptional level follows complex patterns of regulation. J. Cell. Biochem. 70:8-21, 1998. © 1998 Wiley-Liss, Inc.
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  • 177
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    Journal of Cellular Biochemistry 70 (1998), S. 22-28 
    ISSN: 0730-2312
    Keywords: Msx-1 ; Msx-2 ; homeobox ; adhesion ; sorting ; cadherin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Msx-1 and Msx-2 are two closely related homeobox genes expressed in cephalic neural crest tooth buds, the optic cup endocardial cushions, and the developing limb [Hill and Davidson, 1991; Monaghan et al., 1991; Robert et al., 1991]. These sites correspond to regions of active cell segregation and proliferation under the influence of epithelial-mesenchymal cell interactions [Brown et al., 1993; Davidson et al., 1991], suggesting that Msx-1 and Msx-2 regulate cell-cell interactions. We have investigated the potential relationship between expression of the Msh homeobox genes (Msx-1 and Msx-2) and cadherin-mediated cell adhesion and cell sorting. We report that cell lines stably expressing Msx-1 or Msx-2 differentially sort on the basis of Msh gene expression. We demonstrate in vitro that initial cell aggregation involves calcium-dependent adhesion molecules (cadherins) and that Msh genes regulate cadherin-mediated adhesion. These results support the hypothesis that Msh genes play a role in the regulation of cell-cell adhesion and provide a link between the genetic phenomena of homeobox gene expression and cellular events involved in morphogenesis, including cell sorting and proliferation. J. Cell. Biochem. 70:22-28, 1998. © 1998 Wiley-Liss, Inc.
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  • 178
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    Journal of Cellular Biochemistry 70 (1998), S. 29-37 
    ISSN: 0730-2312
    Keywords: small GTPase ; membrane traffic ; vesicles ; transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Eukaryotic cells achieve complexity by compartmentalizing a subset of cellular functions into membrane-bound organelles. Maintaining this high level of cellular organization requires precise regulation of traffic between membranes. This task is accomplished, in part, by rab proteins. How these small GTPases regulate membrane traffic between cellular compartments is not clear. Here we report the characterization of a novel rab GTPase from the soil amoebae Dictyostelium discoideum. The predicted coding sequence of the new rab gene, Dictyostelium rab11b, encodes a protein of 25 kD containing all the structural hallmarks of a rab GTPase. Comparison of the sequence with the GenBank database and cladistic analysis demonstrated Dictyostelium rab11b to be a divergent member of the rab11 branch of rab proteins. Southern analysis revealed the presence of related genes in Dictyostelium. RNAse protection assays showed the Dictyostelium rab11b gene to be expressed at uniform levels throughout growth and development. Gene deletion experiments revealed that Dictyostelium rab11b was not essential for growth or development. Conceivably, the function of rab11b may be redundant with that of related genes in this organism. J. Cell. Biochem. 70:29-37, 1998. © 1998 Wiley-Liss, inc.
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  • 179
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    Journal of Cellular Biochemistry 70 (1998), S. 38-48 
    ISSN: 0730-2312
    Keywords: GABAA receptor ; N-glycosylation ; radioligand binding ; in situ trypsinization ; galactosylation ; mannosylation ; immunoblotting ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of γ-aminobutyric acidA receptor (GABAAR) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 μg/ml of tunicamycin for 24 h blocked the binding of 3H-flunitrazepam and 3H-muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%, respectively. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABAAR immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased to 24% from 34% of control in the presence of the agent, as revealed by trypsinization of cells in situ followed by 3H-flunitrazepam binding. The molecular weight of the receptor subunit protein was lowered around 0.5 kDa after tunicamycin treatment, in accordance with that following N-glycosidase F digestion, indicating the blockade of N-linked glycosylation of GABAAR by tunicamycin. Moreover, intense inhibitions of 91% and 44%, respectively, were detected to the general galactosylation and mannosylation in the tunicamycin-treated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of 3H-sugars and 3H-leucine. Nevertheless, treatment with castanospermine or swainsonine (10 μg/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABAAR α1-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABAAR by producing irregularly glycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N-linked glycosylation may be important for the functional expression and intracellular transport of GABAAR. J. Cell. Biochem. 70:38-48, 1998. © 1998 Wiley-Liss, Inc.
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  • 180
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    Journal of Cellular Biochemistry 70 (1998), S. 60-69 
    ISSN: 0730-2312
    Keywords: opossum kidney cells ; opioid receptors ; actin ; microfilament reorganization ; cell proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recently we identified and characterized opioid binding sites in OK (opossum kidney) cells and observed decreased proliferation of these cells in response to opioids. In the present study we investigated the effects of opioids on the actin cytoskeleton and explored whether their antiproliferative action may relate to alterations in the distribution or the dynamics of actin microfilaments. Exposure of OK cells to the opioids αS1 casomorphin and ethylketocyclazocine resulted in a rapid and substantial actin microfilament reorganization. This was documented by a significant dose-dependent decrease in the amounts of F-actin, determined by measurements of quantitative fluorescence, by immunoblot analysis and by a concomitant increase of the G/total-actin ratio measured by the DNase I inhibition assay. These changes were verified by confocal laser scanning microscopy, which showed marked redistribution of the microfilamentous structures in the presence of the opioids without affecting the organization of microtubules or vimentin intermediate filaments. The effect of opioids on actin polymerization dynamics occurred within 15 min and persisted for at least 2 h, while their restoration to control levels was accomplished 6 h later, indicating a reversible phenomenon. Northern blot analysis showed that the concentration of the actin transcript was unaffected. The addition of diprenorphine, a general opioid antagonist, prevented the effects of opioids on the actin cytoskeleton. The inhibition of OK cell proliferation, induced by ethylketocyclazocine and αS1 casomorphin was partially prevented in the presence of phallacidin, which stabilizes microfilaments. Our findings demonstrate that opioids, acting via kappa 1 binding sites, induce rapidly modifications in the dynamics of actin polymerization, and in the organization of microfilaments in OK cells, which may relate to their antiproliferative effect on these cells. J. Cell. Biochem. 70:60-69, 1998. © 1998 Wiley-Liss, Inc.
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  • 181
    ISSN: 0730-2312
    Keywords: coronary artery ; NO/EDRF ; adenosine ; prostacyclin ; phospholamban ; myosin light chain ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The intracellular mechanisms underlying the action of the endogenous vasodilators such as NO/EDRF, adenosine, and prostacyclin acting through cGMP and cAMP, respectively, are not well understood. One important action of cyclic nucleotides in smooth muscle relaxation is to lower the cytosolic Ca2+ concentration by enhanced sequestration into the sarcoplasmic reticulum. The present study was undertaken to elucidate the potential role of phosphorylation of phospholamban, the regulator of sarcoplasmic reticulum Ca2+ pump, for the control of coronary vascular tone by NO/EDRF, adenosine, and prostacyclin. Phospholamban was identified in pig coronary artery preparations by immunofluorescence microscopy, Western blotting and in vitro phosphorylation. Segments of pig coronary artery, with either intact or denuded endothelium, were precontracted with prostaglandin F2α (PGF2α). In endothelium-denuded preparations 3-morpholinosydnonimine (SIN-1), 5′-N-ethylcarboxiamidoadenosine (NECA), and iloprost (ILO) caused both relaxation and phospholamban phosphorylation with the potency: SIN-1 〉 NECA 〉 ILO. The regulatory myosin light chain was significantly dephosphorylated only by SIN-1. In endothelium-intact pig coronary artery, L-NAME caused additional vasoconstriction and a decrease in phospholamban phosphorylation, while phosphorylation of myosin light chain remained unchanged. An inverse relationship between phospholamban phosphorylation and vessel tone was obtained. Our findings demonstrate significant phospholamban phosphorylation during coronary artery relaxation evoked by NO, prostacyclin, and adenosine receptor activation. Because of the close correlation between phosphorylation of phospholamban and vessel relaxation, we propose that phospholamban phosphorylation is an important mechanism by which endogenous vasodilators, especially endothelial NO/EDRF, control coronary vascular smooth muscle tone. J. Cell. Biochem. 70:49-59, 1998. © 1998 Wiley-Liss, Inc.
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  • 182
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    Journal of Cellular Biochemistry 70 (1998), S. 70-83 
    ISSN: 0730-2312
    Keywords: TGF-β1 ; apoptosis ; growth inhibition ; retina ; endothelial cells ; pericytes ; angiogenesis ; p21waf1/cip1 ; p53 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transforming growth factor-β1 (TGF-β1) regulates a variety of cellular functions. In several types of cells, for example, it acts as a growth inhibitor and an inducer of apoptotic cell death. Although one of the important modulators in retinal vascular development and retinal neovascularization, the effects of TGF-β1 on retinal microvascular cells are not fully defined. We have found that proliferation of both bovine retinal endothelial cells (EC) and pericytes was inhibited by TGF-β1 in a concentration-dependent manner. However, only retinal EC lost viability after exposure to increasing concentrations of TGF-β1 (up to 10 μg/ml) in the presence of 2% fetal bovine serum. Dying EC exhibited the morphological and biochemical characteristics of apoptosis. Fragmented nuclei and chromatin condensation were apparent after staining with the fluorochrome Hoechst 33258 and the reagent ApopTag; moreover, gel electrophoresis of DNA from TGF-β1-treated EC demonstrated degradation of chromatin into the discrete fragments typically associated with apoptosis. The addition of anti-TGF-β1 neutralizing antibody abolished the apoptotic cell death induced by TGF-β1. Because not all the EC in a given culture died after exposure to TGF-β1, we separated the apoptosis-sensitive cells from those resistant to TGF-β1-mediated apoptosis and determined the expression of several proteins associated with this apoptotic pathway. Apoptosis of EC mediated by TGF-β1 was associated with a decreased level of the cyclin-dependent kinase inhibitor p21waf1/cip1, compared with that observed in the apoptosis-resistant cells. In contrast, the translation product of the tumor-suppressor gene p53 was increased in the TGF-β1-treated apoptotic cells. Thus, we propose that p21waf1/cip1 and p53 function in distinct pathways that are protective or permissive, respectively, for the apoptotic signals mediated by TGF-β1. J. Cell. Biochem. 70:70-83, 1998. © 1998 Wiley-Liss, Inc.
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  • 183
    ISSN: 0730-2312
    Keywords: PTHrP ; PTH/PTHrP receptor ; estrogen ; ovariectomy ; kidney ; rat ; in vivo ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The aim of the present study was to test the hypothesis that the decreased renal tubular reabsorption of calcium observed in estrogen deficiency is associated with a local regulation of either PTHrP or PTH/PTHrP receptor genes in the kidney. Rats were randomly sham-operated (S) or ovariectomized receiving either vehicule (OVX) or 4 μg E2/kg/day (OVX+E4) or 40 μg E2/kg/d (OVX+E40) during 14 days using alzet minipumps. Plasma PTH and calcium levels were lower in untreated OVX animals than in all other groups (P 〈 0.01). Plasma PTH was higher in OVX+E40 than in OVX+E4 (P 〈 0.05). PTHrP mRNA expression in the kidney was unaffected by ovariectomy but was increased in OVX+E40 (0.984 ± 0.452 for PTHrP/GAPDH mRNAs expression vs. 0.213 ± 0.078 in sham, P 〈 0.01). PTH/PTHrP receptor mRNA expression and the cAMP response of renal membranes to PTH were unaffected by ovariectomy and estrogen substitution. In conclusion, renal PTHrP and PTH/PTHrP receptor mRNAs are not modified by ovariectomy. However, 17β-estradiol increases renal expression of PTHrP mRNA without evident changes in its receptor expression and function. This may help to explain the pharmacological action of estrogen in the kidney, especially how it prevents the renal leak of calcium in postmenopausal women. J. Cell. Biochem. 70:84-93, 1998. © 1998 Wiley-Liss, Inc.
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  • 184
    ISSN: 0730-2312
    Keywords: steroid hormone receptor ; 1,25-dihydroxyvitamin D3 ; nuclear retention ; DNA-binding ; transcriptional activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA-binding zinc fingers that is similar to well-characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D-responsive element in DNA, in vitro, and hVDR-mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR-specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS-7 cells. Although wild-type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg-49 and Lys-55 contains a novel constitutive nuclear localization signal, RRSMKRK. J. Cell. Biochem. 70:94-109, 1998. © 1998 Wiley-Liss, Inc.
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  • 185
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    Journal of Cellular Biochemistry 70 (1998), S. 110-120 
    ISSN: 0730-2312
    Keywords: myosin heavy chains ; rat heart ; naturally occurring antisense mRNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Analysis of mRNA by Northern blot and reverse transcription-polymerase chain reaction demonstrated the expression of sense and considerable amounts of naturally occurring antisense mRNA for β-myosin heavy chain (MHC) and α-MHC in the neonatal rat heart: antisense MHC mRNA expression of α-MHC and β-MHC was approximately half of the corresponding sense MHC mRNA expression. Using a computational approach, we could identify a reverse Pol II promoter in the β-MHC gene. Both sense and antisense MHC mRNA demonstrated similar sizes of approximately 6,000 bp in the Northern blot. Alpha-MHC antisense mRNA consisted of approximately 3,700 bp of complementary exon sequences and β-MHC consisted of approximately 2,700 bp, suggesting a higher probability of α-MHC mRNA dimerization. Hence, sense mRNA transcripts and protein of α-MHC should exist at different relative levels in the neonatal state. In fact, the relative proportion of α-MHC was 52.0 ± 2.6% on the sense mRNA but only 36.3 ± 1.8% on the protein level. Because of its high abundance in the heart, we suggest that in the neonatal heart naturally occurring antisense mRNA may play a role in the regulation of MHC expression and, therefore, in the control of the energetical and contractile behaviour of the heart. J. Cell. Biochem. 70:110-120, 1998. © 1998 Wiley-Liss, Inc.
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  • 186
    ISSN: 0730-2312
    Keywords: giant cell tumor of bone ; MCP-1 ; TGF-β ; CD68+ ; chemotaxis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Giant cell tumor of bone (GCT) is one of a few neoplasms in which the macrophage/osteoclast precursor cells and osteoclast-like giant cells infiltrate the tumor mass. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemotactic factor specific for monocytes. In search of relevant cytokines that may enhance the recruitment of these reactive cells, we evaluated the localization and regulation of MCP-1 mRNA and protein in GCT by using Northern blot analysis, in situ hybridization and immunohistochemistry. We also determined whether conditioned medium obtained from GCT cultures can recruit human peripheral blood monocytes (CD68+) in an in vitro chemotactic assay. Using Northern blot analysis, we detected the specific gene transcript for MCP-1 in all GCT samples tested. In situ hybridization and immunohistochemistry revealed that both MCP-1 gene transcript and protein were consistently present in the cytoplasm of stromal-like tumor cells of GCT. Treatment of mononuclear cells from GCT at third passage with TGF-β1 for 24 h increased the level of MCP-1 mRNA in a dose-dependent manner, with the maximum effect at 1 ng/ml. Conditioned media from GCT cultures promoted the chemotactic migration of CD68+ peripheral monocytes, an activity which was abolished by the addition of MCP-1 antibody to the conditioned medium. Thus, the results of this study suggest that recruitment of CD68+ macrophage-like cells may be due to the production MCP-1 by stromal-like tumor cells. These CD68+ cells may originate from peripheral blood and could have the capability of further differentiating into osteoclasts in the tumor. J. Cell. Biochem. 70:121-129, 1998. © 1998 Wiley-Liss, Inc.
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  • 187
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    Journal of Cellular Biochemistry 70 (1998), S. 157-158 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
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  • 188
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    Journal of Cellular Biochemistry 70 (1998), S. 141-155 
    ISSN: 0730-2312
    Keywords: endothelial cell ; tyrosine phosphatase ; vanadate ; permeability ; MLCK ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The involvement of tyrosine protein phosphorylation in the regulation of endothelial cell (EC) contraction and barrier function is poorly understood. We have previously shown that myosin light chain (MLC) phosphorylation catalyzed by a novel 214 kDa EC myosin light chain kinase (MLCK) isoform is a key event in EC contraction and barrier dysfunction [Garcia et al. (1995): J Cell Physiol 163:510-522; Garcia et al. (1997): Am J Respir Cell Mol Biol 16:487-491]. In this study, we tested the hypothesis that tyrosine phosphatases participate in the regulation of EC contraction and barrier function via modulation of MLCK activity. The tyrosine phosphatase inhibitor, sodium orthovanadate (vanadate), significantly decreased electrical resistance across bovine EC monolayers and increased albumin permeability consistent with EC barrier impairment. Vanadate significantly increased EC MLC phosphorylation in a time-dependent manner (maximal increase observed at 10 min) and augmented both the MLC phosphorylation and permeability responses produced by thrombin, an agonist which rapidly increases tyrosine kinase activities. The vanadate-mediated increase in MLC phosphorylation was not associated with alterations in either phosphorylase A Ser/Thr phosphatase activities or in cytosolic [Ca2+] but was strongly associated with significant increases in EC MLCK phosphotyrosine content. These data suggest that tyrosine phosphatase activities may participate in EC contractile and barrier responses via the regulation of the tyrosine phosphorylation status of EC MLCK. J. Cell. Biochem. 70:141-155, 1998. © 1998 Wiley-Liss, Inc.
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  • 189
    ISSN: 0730-2312
    Keywords: signal transduction ; chromatin structure ; cytology ; histones ; metastasis ; Ras ; MAPKK ; NIH3T3 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: An altered nuclear morphology has been previously noted in association with Ras activation, but little is known about the structural basis, functional significance, signaling pathway, or reproducibility of any such change. We first tested the reproducibility of Ras-associated nuclear change in a series of rodent fibroblast cell lines. After independently developing criteria for recognizing Ras-associated nuclear change in a Papanicolaou stained test cell line with an inducible H(T24)-Ras oncogene, two cytopathologists blindly and independently assessed 17 other cell lines. If the cell lines showed Ras-associated nuclear change, a rank order of increasing nuclear change was independently scored. Ras-associated nuclear changes were identified in v-Fes, v-Src, v-Mos, v-Raf, and five of five H(T24)-Ras transfectants consisting of a change from a flattened, occasionally undulating nuclear shape to a more rigid spherical shape and a change from a finely textured to a coarse heterochromatic appearance. Absent or minimal changes were scored in six control cell lines. The two cytopathologists' independent morphologic rank orders were similar (P〈 .0002). The mitogen signaling pathway per se does not appear to transduce the change since no morphologic alterations were identified in cell lines with activations of downstream components of this pathway - MAPKK or c-Myc - and the rank orders did not correlate with markers of mitotic rate (P 〉 .11). The rank order correlated closely with metastatic potential (P 〈 .0014 and P 〈 .0003) but not with histone H1 composition or global nuclease sensitivity. Based on published studies of five of the cell lines, there may be a correlation between increases in certain nuclear matrix proteins and the Ras-associated nuclear change. J. Cell. Biochem. 70:130-140, 1998. © 1998 Wiley-Liss, Inc.
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  • 190
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    Journal of Cellular Biochemistry 70 (1998), S. 159-171 
    ISSN: 0730-2312
    Keywords: nucleus ; nuclear domain ; genome ; nucleolus ; coiled body ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It is becoming clear that the cell nucleus is not only organized in domains but that these domains are also organized relative to each other and to the genome. Specific nuclear domains, enriched in different proteins and RNAs, are often found next to each other and next to specific gene loci. Several lines of investigation suggest that nuclear domains are involved in facilitating or regulating gene expression. The emerging view is that the spatial relationship between different domains and genes on different chromosomes, as found in the nucleolus, is a common organizational principle in the nucleus, to allow an efficient and controlled synthesis and processing of a range of gene transcripts. J. Cell. Biochem. 70:159-171. © 1998 Wiley-Liss, Inc.
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  • 191
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    Journal of Cellular Biochemistry 70 (1998), S. 172-180 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; nuclear structure ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172-180, 1998. © 1998 Wiley-Liss, Inc.
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  • 192
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    Journal of Cellular Biochemistry 70 (1998), S. 181-192 
    ISSN: 0730-2312
    Keywords: coiled bodies (CBs) ; gems ; p80 coilin ; RNPs ; RNA processing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Coiled bodies (CBs) are nuclear organelles whose morphology and composition have been conserved from plants to animals. They are highly enriched in components of three different RNA processing pathways. Small nuclear RNAs (snRNAs) involved in pre-mRNA splicing, rRNA processing, and histone mRNA 3′ end maturation all take up residence in CBs. However, CB function(s) remain obscure. This review will focus on recent developments in several aspects of CB structure and function, including exciting new results on their twin organelles, called gems. In particular, the reader will be introduced to a novel hypothesis called the “salmon theory of snRNP biogenesis.” Questions arising from and experiments necessary to test this hypothesis will be discussed. J. Cell. Biochem. 70:181-192, 1998. © 1998 Wiley-Liss, Inc.
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  • 193
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    Journal of Cellular Biochemistry 70 (1998), S. 193-199 
    ISSN: 0730-2312
    Keywords: parathyroid hormone-related peptide ; nucleus ; nucleolus ; intracrine actions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It is becoming increasingly apparent that parathyroid hormone-related peptide (PTHrP) modulates cellular function in a dual mode of action: first, by binding and activating its cognate cell surface G-protein-coupled receptor and, second, by direct intracellular effects following translocation to the nucleus and/or nucleolus of the target cell. Little is presently known about the mechanisms and events that determine the timing and degree of PTHrP nuclear translocation or the role it may serve in normal or dysregulated cellular function. Clarifying the nuclear actions of PTHrP would add significantly to our present understanding of this protein as a signaling molecule during embryonic development and as an oncoprotein whose expression in many tumors correlates with increased tumor aggressiveness and propensity for metastasis. J. Cell. Biochem. 70:193-199, 1998. © 1998 Wiley-Liss, Inc.
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  • 194
    ISSN: 0730-2312
    Keywords: gene expression ; AML/CBF transcription factors ; nuclear matrix ; cancer ; nuclear domains ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. In this article we focus on the concept that association of genes and cognate transcription factors with the nuclear matrix may support the formation and/or activities of nuclear domains that facilitate transcriptional regulation. Several lines of evidence are consistent with the concept that association of transcription factors with the nuclear matrix may be obligatory for fidelity of gene expression and maximal transcriptional activity. The identification of specific regions of transcription factors that are responsible for intranuclear trafficking to nuclear matrix-associated sites that support transcription, reinforces the linkage of nuclear structure to regulation of genes. CBFA2/AML-1 and CBFA1/AML-3 provide paradigms for directing gene regulatory factors to RNA polymerase II containing foci within the nucleus. The implications of modifications in the intranuclear trafficking of transcription factors for developmental and tissue-specific control, as well as for aberrations in gene expression that are associated with cancer and neurological disorders, are addressed. J. Cell. Biochem. 70:200-212, 1998. © 1998 Wiley-Liss, Inc.
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  • 195
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    Journal of Cellular Biochemistry 70 (1998), S. 213-221 
    ISSN: 0730-2312
    Keywords: transcription ; nucleus ; cell architecture ; nuclear matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: After many years of reductionistic approaches to characterize molecular mechanisms involved in transcription, the number of factors recognized to take part in this process has increased remarkably and continues to grow. When considering posttranslational modifications in conjunction with the large number of factors involved in modulating the activity of transcription complex components, the overall intricacy becomes staggering. After two decades of intensive molecular investigations, there has been a concerted effort to integrate these findings with cellular approaches to understand transcription on a more global level. This sort of reasoning actually revisits studies of approximately 20 years ago that considered the functional consequences of steroid receptor association with nuclear structure. With an abundance of new molecular probes and increasingly powerful instruments to detect them in fixed and, more recently, live cells, the issue of functional subnuclear organization is receiving increased attention. In this report, we focus on advances in characterizing the functional significance of transcription factor association with the nucleoskeleton. In particular, we consider recent biochemical and “molecular morphology” data that point to the importance of dynamic spatial and solubility partitioning of gene regulators with nuclear architecture. J. Cell. Biochem. 70:213-221, 1998. © 1998 Wiley-Liss, Inc.
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  • 196
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    Journal of Cellular Biochemistry 70 (1998), S. 222-230 
    ISSN: 0730-2312
    Keywords: functional organization of the nucleus ; nucleolus ; speckled compartment ; targeting sequence ; DNA replication ; RNA splicing ; nuclear matrix ; cell cycle ; DNA methyltransferase ; DNA ligase I ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Though there are no separating membranes within the nucleus, different factors are often concentrated at sites where their respective function is required, a phenomenum referred to as functional organization of the nucleus. How is then this organization achieved and how are the different metabolic processes integrated in the nucleus? One emerging principle was revealed by the identification of protein domains that, though not involved in catalysis, regulate enzyme activity at a higher order level by targeting enzymes to the right place at the right time. These targeting sequences constitute an assembly code for nuclear ‘protein factories,’ which ensure the extremely high efficiency and accuracy needed in a complex and competitive environment as the living mammalian cell. J. Cell. Biochem. 70:222- 230, 1998. © 1998 Wiley-Liss, Inc.
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  • 197
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    Journal of Cellular Biochemistry 70 (1998), S. 231-239 
    ISSN: 0730-2312
    Keywords: nuclear pore complexes ; nuclear localization signals ; nuclear export signals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of karyopherin α and β1, that docks via β1 the NLS-protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS-protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the karyopherin β family have been discovered. Karyopherin β2 mediates import of mRNA binding proteins, whereas karyopherin β3 and β4 mediate import of a set of ribosomal proteins. Two other β karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine-rich nuclear export signals (NES) and reexport of karyopherin α, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of karyopherin β family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the karyopherin β superfamily and to characterize the molecular mechanisms of translocation of karyopherins together with their cargoes through the NPC. J. Cell. Biochem. 70:231-239, 1998.© 1998 Wiley-Liss, Inc.
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  • 198
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    Journal of Cellular Biochemistry 70 (1998), S. 240-251 
    ISSN: 0730-2312
    Keywords: actin ; actin-like proteins ; lamin ; nuclear matrix ; perinuclear actin shells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Perinuclear actin shells have been reported in a variety of organisms. The shells have been identified by staining perinuclear material with fluorescently-labelled phalloidin, but have not been localized to a specific subcellular compartment at the ultrastructural level. We show here that the shells of 3T3 cells lie in the peripheral nuclear matrix. Nuclear shells and matrix actin in other parts of the nucleus are not usually detected by immunohistochemical staining because they are inaccessible to antibodies or to phalloidin. Immunohistochemical detection of nuclear actin is only possible during its deposition at the end of mitosis, or in interphase nuclei that have been extracted with detergent, digested with nucleases and washed with high salt buffers. J. Cell. Biochem. 70:240-251, 1998. © 1998 Wiley-Liss, Inc.
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  • 199
    ISSN: 0730-2312
    Keywords: insulin ; insulin receptor ; breast cancer cells ; insulin receptor substrate 1 ; phosphatidylinositol-3-kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol-3-kinase (PI3-K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin-stimulated cell growth in human breast cancer, we measured insulin-stimulable PI3-K activity in a non-transformed breast epithelial cell line, MCF-10A, and in two malignantly transformed cell lines, ZR-75-1 and MDA-MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA-MB157 and MCF-10A cells, insulin stimulated PI3-K activity three- to fourfold. In ZR-75-1 cells, however, insulin did not stimulate PI3-K activity. In ZR-75-1 cells PI3-K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3-K and downstream of the insulin receptor. Next, we studied insulin receptor substrate-1 (IRS-1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3-K. In ZR-75-1 cells, there were reduced levels of protein for IRS-1. In these cells, both Shc tyrosine phosphorylation and mitogen-activated protein kinase (MAP-K) activity were increased by the insulin receptor (indicating that the p21ras pathway may account for insulin-stimulated cell growth in ZR-75-1 cells).The PI3-K inhibitor LY294002 (50 μM) reduced insulin-stimulated growth in MCF-10A and MDA-MB157 cell lines, whereas it did not modify insulin effect on ZR-75-1 cell growth. The MAP-K/Erk (MEK) inhibitor PD98059 (50 μM) consistently reduced insulin-dependent growth in all three cell lines.Taken together, these data suggest that in breast cancer cells insulin may stimulate cell growth via PI3-K-dependent or-independent pathways. J. Cell. Biochem. 70:268-280, 1998. © 1998 Wiley-Liss, Inc.
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  • 200
    ISSN: 0730-2312
    Keywords: lymphocyte ; monocyte ; cell line ; cell culture ; microgravity ; experiment development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252-267, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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