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  • 1
    Electronic Resource
    Electronic Resource
    Platelet talin is phosphorylated by calyculin A (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 120-126 
    Details
    ISSN: 0730-2312
    Keywords: protein phosphatase ; calyculin A ; platelet ; talin ; phosphorylation ; phosphoamino acid analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240570112
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  • 2
    Electronic Resource
    Electronic Resource
    TGF-β regulation of nuclear proto-oncogenes and TGF-β gene expression in normal human osteoblast-like cells (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 52-61 
    Details
    ISSN: 0730-2312
    Keywords: TGF-β ; c-fos ; jun-B ; promoter regulation ; osteoblasts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transforming growth factor-β (TGF-β) is present in high levels in bone and plays an important role in osteoblast growth and differentiation. In order to dissect the molecular mechanisms of action of TGF-β on osteoblasts, the effects of TGF-β on the steady state mRNA levels of c-fos, c-jun, and jun-B proto-oncogenes on normal human osteoblast-like cells (hOB) and a transformed human osteoblast cell line (MG-63) were measured. Treatment of hOBs with 2 ng/ml of TGF-β1 resulted in a rapid increase in c-fos mRNA levels as early as 15 min post-treatment. A maximum (10-fold) increase was observed at 30 min after TGF-β treatment followed by a decrease to control values. Similar responses were measured whether the cells were rapidly proliferating or quiescent. TGF-β1 induced jun-B mRNA levels more gradually with steady increase initially observed at 30 min and a maximum induction measured at 2 h post-TGF-β treatment. In contrast, TGF-β treatment caused a time dependent decrease in the c-jun mRNA levels, an opposite pattern to that of jun-B mRNA. Treatment of hOBs with TGF-β1 in the presence of actinomycin-D abolished TGF-β1 induction of c-fos mRNA, suggesting that TGF-β action is mediated via transcription. In the presence of cycloheximide, TGF-β causes super-induction of c-fos mRNA at 30 min, indicating that the c-fos expression by TGF-β is independent of new protein synthesis. Further, transfection of 3 kb upstream region of jun-B promoter linked to a CAT reporter gene into ROS 17/2.8 cells was sufficient to be regulated by TGF-β1. Interestingly, TGF-β treatment also increased the mRNA levels of TGF-β1 itself at 4 h post TGF-β treatment, with a maximum increase observed at 14 h of treatment. TGF-β1 treatment for 30 min were sufficient to cause a delayed increase in TGF-β protein secretion within 24 h. These data support that TGF-β has major effects on hOB cell proto-oncogene expression and that the nuclear proto-oncogenes respond as rapid, early genes in a cascade model of hormone action.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240570107
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  • 3
    Electronic Resource
    Electronic Resource
    Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO42- uptake and expression of a band 3 like protein in the marine sponge, Microciona prolifera (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 71-89 
    Details
    ISSN: 0730-2312
    Keywords: proteoglycan secretion ; aggregation ; AP ; SDS-PAGE ; sulfate deprived cells ; cell motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: (1) impairment of aggregation, (2) loss of cell movements, (3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of 35SO42- uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in 35SO42- uptake and incorporation which could be greatly augmented in the presence of Ca2+/Mg2+. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa 35SO42- sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.
    Additional Material: 10 Ill.
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    URL: http://dx.doi.org/10.1002/jcb.240570109
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  • 4
    Electronic Resource
    Electronic Resource
    Proximal promoter binding protein contributes to developmental, tissue-restricted expression of the rat osteocalcin gene (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 90-100 
    Details
    ISSN: 0730-2312
    Keywords: osteocalcin ; homeodomain protein ; osteoblasts ; transcriptional regulation ; bone specific ; developmental ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteocalcin is a 6 kD tissue-specific calcium binding protein associated with the bone extracellular matrix. The osteocalcin gene is developmentally expressed in postproliferative rat osteoblasts with regulation at least in part at the transcriptional level. Multiple, basal promoter and enhancer elements which control transcriptional activity in response to physiological mediators, including steroid hormones, have been identified in the modularly organized osteocalcin gene promoter. The osteocalcin box (OC box) is a highly conserved basal regulatory element residing between nucleotides -99 and -76 of the proximal promoter. We recently established by in vivo competition analysis that protein interactions at the CCAAT motif, which is the central core of the rat OC box, are required for support of basal transcription [Heinrichs et al. J Cell Biochem 53:240-250, 1993]. In this study, by the combined utilization of electrophoretic mobility shift analysis, UV cross linking, and DNA affinity chromatography, we have identified a protein that binds to the rat OC box. Results are presented that support involvement of the OC box-binding protein in regulating selective expression of the osteocalcin gene during differentiation of the rat osteoblast phenotype and suggest that this protein is tissue restricted.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240570110
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  • 5
    Electronic Resource
    Electronic Resource
    Generation of phosphorylcholine as an essential event in the activation of Raf-1 and MAP-kinases in growth factors-induced mitogenic stimulation (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 141-149 
    Details
    ISSN: 0730-2312
    Keywords: Growth factors ; cell growth ; phospholipase D ; hemicholinium-3 ; phosphorylcholine ; choline kinase ; Raf-1 ; MAP kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cell proliferation is regulated by an appropriate combination of intracellular signals involving activation of kinases and the generation of phospholipid metabolites. We report here that growth factors induce a biphasic generation of phosphorylcholine (PCho) in quiescent NIH 3T3 cells, resulting in an early and transient increase at 100 s and a larger and sustained increase after 3 h of stimulation. Generation of PCho at both early and late times of growth factors stimulation results from the consecutive activation of phospholipase D (PLD) and choline kinase (ChoK). Production of PCho by specific growth factors seems an essential requirement for the early signals associated to activation of Raf-1 and MAP kinases, since blockage of choline kinase completely inhibited activation of Raf-1 and MAP kinases by PDGF or FGF. Both the transient early increase and the late sustained increase in PCho are required for the induction of DNA-synthesis, besides completion of the activation of the serine/threonine kinases cascade. Thus, our results strongly suggest that generation of PCho by the PLD/choline kinase pathway is one of the critical steps in regulating cell growth in NIH 3T3 stimulated by growth factors.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/jcb.240570114
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  • 6
    Electronic Resource
    Electronic Resource
    Integrity of intermediate filaments is associated with the development of acquired thermotolerance in 9L rat brain tumor cells (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 150-162 
    Details
    ISSN: 0730-2312
    Keywords: thermotolerance ; heat-shock response ; cytoskeletal systems ; vimentin ; HSC70 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Withangulatin A (WA), a newly discovered withanolide isolated from an antitumor Chinese herb, has been shown to be a vimentin intermediate filament-targeting drug by using immunofluorescence microscopy. Together with cytochalasin D and colchicine, these drugs were employed to investigate the importance of vimentin intermediate filaments, actin filaments, and microtubules in the development of acquired thermotolerance in 9L rat brain tumor cells treated at 45°C for 15 min (priming heat-shock). Acquired thermotolerance was abrogated in cells incubated with WA before the priming heat-shock but it could be detected in cells treated with WA after the priming heat-shock. In contrast, cytochalasin D and colchicine do not interfere with the development of thermotolerance at all. The intracellular localizations of vimentin and the constitutive heat-shock protein70 (HSC70) in treated cells were examined by using immunofluorescence microscopy and detergent-extractability studies. In cells treated with WA before the priming heat-shock, vimentin IFs were tightly aggregated around the nucleus and unable to return to their normal organization after a recovery under normal growing conditions. In contrast, the IF network in cells treated with WA after the priming heat-shock was able to reorganize into filamentous form after a recovery period, a behavior similar to that of the cells treated with heat-shock only. HSC70 was found to be co-localized with vimentin during these changes. It is suggested that the integrity of intermediate filaments is important for the development of thermotolerance and that HSC70 may be involved in this process by stabilizing the intermediate filaments through direct or indirect binding.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/jcb.240570115
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  • 7
    Electronic Resource
    Electronic Resource
    Rodent myoblast interactions with laminin require cell surface glycoconjugates but not laminin glycosyl groups (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 163-172 
    Details
    ISSN: 0730-2312
    Keywords: glycosylation ; laminin ; myoblasts ; lectins ; ConA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Laminin glycosyl groups are necessary for the spreading of murine melanoma cells which become attached to this glycoprotein. Laminin has been implicated in myogenesis but the potential role of its glycosyl groups in this process has not been examined. In this study we report the effects of the carbohydrate moieties of laminin on myoblast adhesion, spreading, and differentiation. Unglycosylated laminin from tunicamycin-treated cultures of a mouse cell line, M1536 B3, was used in the experiments. Glycosylated laminin from a murine tumor and from cultures of M1563 B3 cells served as controls. Cell binding experiments with C2C12 mouse myoblasts showed that the cells preferred a laminin-coated surface, compared to the uncoated plastic surface (nontissue culture wells). Myoblasts did not distinguish between glycosylated and unglycosylated laminin substrates. Both glycosylated and unglycosylated forms of laminin promoted myoblast growth and differentiation. In contrast, cells on uncoated plastic surfaces grew very slowly and did not further differentiate. The L6 rat myoblast response to glycosylated and unglycosylated laminin was the same. These results indicate that although rodent myoblasts in culture require a laminin substratum for spreading, growth, and differentiation on a proprietary plastic surface, laminin carbohydrates are not implicated in those cellular responses. In contrast, parallel studies using the lectin, Con A, indicate that cell surface glycoconjugates of myoblasts are implicated in the response of these cells to a laminin substratum.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240570116
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  • 8
    Electronic Resource
    Electronic Resource
    Heparan sulfate primed on β-D-xylosides restores binding of basic fibroblast growth factor (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 173-184 
    Details
    ISSN: 0730-2312
    Keywords: heparin ; proteoglycans ; glycosaminoglycans ; receptor-binding ; heparinase ; chinese hamster ovary cell mutants ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA-745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure of pgsA-745 cells to β-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell sulfate. Restoration of bFGF binding correlated with the ability of the β-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10-30 μM) of estradiol-β-D-xyloside and naphthyl-β-D-xyloside, but not by cis/trans-decahydro-2-naphthyl-β-D-xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5-1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240570202
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  • 9
    Electronic Resource
    Electronic Resource
    In vivo occupancy of histone gene proximal promoter elements reflects gene copy number-dependent titratable transactivation factors and cross-species compatibility of regulatory sequences (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 191-207 
    Details
    ISSN: 0730-2312
    Keywords: histone gene transcription ; chromosome ; H4 gene ; C127 cell ; titratable transcription factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To assess systematically the structural and functional aspects of histone gene transcription within a chromosomal context, we stably integrated an extensive set of human histone H4 gene constructs into mouse C127 cells. Levels of expression were determined by S1 nuclease protection assays for multiple mouse monoclonal cell lines containing these human H4 genes. For each cell line, we quantitated the number of integrated human H4 genes by Southern blot analysis. The results indicate that the expression of the human H4 gene is in part copy number dependent at low gene dosages. However, the level of expression varies among different cell lines containing similar numbers of copies of the same H4 gene construct. This result suggests that position-dependent chromosomal integration effects contribute to H4 gene transcription, consistent with the roles of long-range gene organization and nuclear architecture in gene regulation. At high copy number, the level of human H4 gene expression per copy decreased, and endogenous mouse H4 mRNA levels were also reduced. Furthermore, in vivo occupancy at the human H4 gene immediate 5′ regulatory elements, as defined by genomic fingerprinting, showed copy number-dependent protein/DNA interactions. Hence, human and mouse H4 genes compete for titratable transcription factors in a cellular environment. Taken together, these results indicate cross-species compatibility and suggest limited representation in vivo of the factors involved in regulating histone H4 gene transcription.
    Additional Material: 12 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240570204
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  • 10
    Electronic Resource
    Electronic Resource
    Early detection and screening for ovarian cancer (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 233-237 
    Details
    ISSN: 0730-2312
    Keywords: CA-125 ; ovarian cancer screening ; tumor markers ; UGP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ovarian cancer is associated with potmenopausal women on North Ameican or European descent, nulliparous women, and women with a first-degree relative with an epithelial overaina cancer. Methods for early detection of ovarian cancer are the pelvic examination, ultrasound techniques, and CA-125 monitoring, none of which are highly sensitive or specific for the disease. At the Yale-New Haven Medical Center, first-degree relatives of women with epithelial ovarian cancer were invited to participate in an intense ovarian cancer screening program consisting of tumor markers, endovaginal ultrasound and color Doppler flow studies, and physical examinations performed in a serial fashion. The false-postive rate for the tumor markers varied from 2 to 9% at initial evaluation of the first 247 participants. Endovaginal ultrasound and color Doppler flow techniques were used to evaluate 326 ovaries in 169 womens. Resistive indices 〈 0.5 were present in 26 ovaries (8.4%) and peak systolic velocities 〉 30 cm/sec occurred in 7 ovaries (2.3%). To date, four breast cancers have been detected, three cervical intraepithelial neoplasias have been identified, and three atypical adenomatous hyperplasias were diagnosed. No epithelial ovarian cancer was found. Isolated screening for ovarian cancer even in high-risk womed is not cost effective. Women screened for ovarian cancer should also be evaluated for cancers of the breast, cervix, colon rectum and endometrium. Isolated abnormal screening test values are not an indication for surgery.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590932
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  • 11
    Electronic Resource
    Electronic Resource
    Screening for ovarian cancer: What are the optimal surrogate endpoints for clinical trials? (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 227-232 
    Details
    ISSN: 0730-2312
    Keywords: Color doppler imaging ; HER-2/neu oncogene ; ovarian cancer screening ; ultrasound ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The inability to identify relevant markers for presymptomatic screening in early stage or “preinvasive” ovarian cancer had plagued investigators and clinicians facing the problems of early detection. The characteristic late stage of disease at initial presentation has hindered our understanding of the biologic progression and stepwise molecular alterations that result in ovarian carcinoma. To date, most screening studies have focused on identifying early anatomic changes using ultrasound or fluctuations in serum biomarkers such as CA-125. These screening methodologies have proven inadequate in both sensitivity and specificity for early stage ovarian cancer detection.Molecular analysis of ovarian carcinomas has revealed alterations in oncogenes and tumor suppressor genes associated with these tumors. The HER-2/neu oncogene, a member of the epidermal growth factor family, is amplified or overexpressed in approximately 25-30% of ovarian carcinomas. Significant data substantiate an important role for HER-2/neu in the pathophysiology of ovarian cancer. While potentially an attractive surrogate endpoint biomarker (SEB), serum HER-2/neu levels have not proven to be a useful screening modality.In response to the urgent need for improved early detection for ovarian cancer, our current research efforts include differential hybridization studies between normal and malignant ovarian epithelium to define potentially unique ovarian cancer antigens which may ultimately have clinical utility; defining physical alterations that occur in malignant ovarian tissues using implanted telemetry systems; studies using positron emission tomography to detect changes in glucose metabolism between normal and malignant ovarian tissues; and screening studies using a 3-dimensional ultarsound unit to improve the accuracy of this technique in recognizing early neoplastic changes. By taking diverse approaches to tackle this problem, an improved understanding of ovarian carcinogenesis should translate into the identification of appropriate SEBs for early detection.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590931
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  • 12
    Electronic Resource
    Electronic Resource
    Signal transduction in plants (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 469-510 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590610
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  • 13
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    Masthead (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995) 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590701
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  • 14
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    Frontiers of NMR in molecular biology - IV (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 13-81 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590703
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  • 15
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    Epigenetic regulation of transcription (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 149-177 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590706
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  • 16
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    HIV pathogenesis (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 179-247 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590707
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  • 17
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    Evolutionary conservation of a genetic pathway of programmed cell death (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 4-11 
    Details
    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: Genetic analysis of programmed cell death in Caenorhabditis elegans has led to the identification of 13 genes that constitute a developmental pathway of programmed cell death. Two of the three key genes in this pathway, ced-9, a cell death suppressor, and ced-3, a cell death inducer, were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1β converting enzyme, respectively. These results suggest that the genetic pathway of programmed cell death may be evolutionarily conserved from worms to mammals. © 1996 Wiley-Liss, Inc.
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  • 18
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    Cofractionation of hela cell replication proteins with Ors-binding activity (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 221-236 
    Details
    ISSN: 0730-2312
    Keywords: ors ; replication origin ; replication proteins ; purification ; HeLa cells ; in vitro replication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases α and δ, topoisomerase II, and replication protein A, (RP-A).
    Additional Material: 11 Ill.
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    URL: http://dx.doi.org/10.1002/jcb.240580211
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  • 19
    Electronic Resource
    Electronic Resource
    Masthead (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995) 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240580301
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    Oncogenes: 20 Years later (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 1-86 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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    URL: http://dx.doi.org/10.1002/jcb.240591002
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    Bacterial chromosomes (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 87-123 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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    URL: http://dx.doi.org/10.1002/jcb.240591003
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    Plant cell biology: Mechanisms, molecular machinery, signals and pathways (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 125-161 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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    URL: http://dx.doi.org/10.1002/jcb.240591004
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    Molecular toxicology (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 181-198 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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    URL: http://dx.doi.org/10.1002/jcb.240591006
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    Molecular aspects of viral immunity (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 269-324 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240591009
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    Genetic networks (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 325-347 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240591010
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    Mucosal immunity: New strategies for protection against viral and bacterial pathogens (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 231-267 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240591008
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    Cancer cell invasion and motility (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 1-31 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590502
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    Molecular mechanisms in tuberculosis (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 55-98 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590504
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    Function of the cytoskeletion in cell growth, organization and differentiation (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 117-139 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590506
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    Synapse formation and function: The neuromuscular junction and central nervous system II (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 165-183 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590508
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    Dendritic cells: Antigen presenting cells of T and B lymphocytes (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 1-36 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590602
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    Environmental biotechnology (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 37-53 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Gene therapy and molecular medicine (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 353-434 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Frontiers of plant morphogenesis (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 435-467 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590609
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    Science and engineering of immunoprotected cell transplants (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 1-11 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590702
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    The molecular and cellular basis of human neurodegenerative disease (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 83-113 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590704
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    The nuclear matrix: Involvement in replication, transcription gene splicing and cellular regulation (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 115-148 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590705
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    Sexually transmitted diseases in the HIV era (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 249-259 
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    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    URL: http://dx.doi.org/10.1002/jcb.240590708
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    BCL-2, a novel regulator of apoptosis (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 12-17 
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    ISSN: 0730-2312
    Keywords: bcl-2 gene ; localization ; apoptosis ; antioxidants ; oxidative stress ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The bcl-2 gene has a unique function among mammalian oncogenes as a negative regulator of apoptosis. Its expression pattern in embryonic and adult tissues is consistent with a role in maintaining in vivo survival of specific cell types.The biochemical function of bcl-2 is unknown, but its localization to mitochondrial and microsomal membranes suggests several possibilities, bcl-2 is protective against oxidative stress in mammalian cells and can be replaced by antioxidants in a factor-deprivation model of apoptosis. These results are consistent with a model of apoptotic death involving oxidative stress in a central pathway.The recent discovery of several bcl-2-related genes, some of which also inhibit apoptosis and others that unexpectedly promote apoptosis, has shed new light on several aspects of bcl-2 action. © 1996 Wiley-Liss, Inc.
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    Regulation of apoptosis by oncogenes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 33-38 
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    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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    BCL-2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 23-32 
    Details
    ISSN: 0730-2312
    Keywords: BCL-2 gene ; Bcl-2 protein ; homologs ; homo- and heterotypic dimers ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The BCL-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL-2 gene expression and cause inappropriately high levels of Bcl-2 protein production. Expression of the BCL-2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL-2 gene expression in some tissues. Bcl-2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl-2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl-2 protein. Many of these Bcl-2 family proteins can interact through formation of homo- and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl-2 and that can modulate apoptosis. These protein-protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases. © 1996 Wiley-Liss, Inc.
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    Nuclear import of protein kinases and cyclins (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 61-82 
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    ISSN: 0730-2312
    Keywords: protein kinases ; cyclins ; nuclear import ; NLS ; acidic domains ; cell cycle ; phosphatases ; p34cdc2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Karyophilic and acidic clusters were found in most nonmembrane serine/threonine protein kinases whose primary structure was examined. These karyophilic clusters might mediate the anchoring of the kinase molecules to transporter proteins for their regulated nuclear import and might constitute the nuclear localization signals (NLS) of the kinase molecules. In contrast to protein transcription factors that are exclusively nuclear possessing strong karyophilic peptides composed of at least four arginines (R) and lysines (K) within an hexapeptide flanked by proline and glycine helix-breakers, protein kinases often contain one histidine and three K + R residues; this is proposed to specify a weak NLS structure resulting in the nuclear import of a fraction of the total cytoplasmic kinase molecules as well as in their weak retention in the different ionic strength nuclear environment. Putative NLS peptides in protein kinases may also contain hydrophobic or bulky aromatic amino acids proposed to further diminish their capacity to act as strong NLS. Most kinases lacking karyophilic clusters (c-Mos, v-Mos, sea star MAP, and yeast KIN28, SRA1, SRA3, TPK1, TPK2) also lack acidic clusters, which is in contrast to most kinases containing both acidic and karyophilic peptides; this and the presence of R/K clusters in the transporter proteins supports a role of acidic clusters on kinases in nuclear import. Cyclins B lack karyophilic signals and are proposed to be imported into nuclei via their association with Cdc2. © 1996 Wiley-Liss, Inc.
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    Calphostin C induces tyrosine dephosphorylation/inactivation of protein kinase Fa/GSK-3α in a pathway independent of tumor promoter phorbol ester-mediated down-regulation of protein kinase C (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 121-129 
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    ISSN: 0730-2312
    Keywords: protein kinase FA/GSK-3α ; PKC inhibition ; calphostin C ; down-regulation ; carcinoma dedifferentiation/progression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The signal transduction mechanism of protein kinase FA/GSK-3α by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase Fa/GSK-3α could be tyrosine-dephosphorylated and inactivated to ∼ 10% of control in a concentration-dependent manner by 0.1-10 μM calphostin C (IC50, ∼ 1 μM), as demonstrated by immunoprecipitation of kinase Fa/GSK-3α from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase Fa/GSK-3α immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 μM calphostin C (IC50, ∼ 0.05 μM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase Fa/GSK-3α, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase Fa/GSK-3α. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase Fa/GSK-3α in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase Fa/GSK-3α is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA/GSK-3α in tumor cells. © 1996 Wiley-Liss, Inc.
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    Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 424-435 
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    ISSN: 0730-2312
    Keywords: Key words ; osteoclast ; focal adhesion kinase ; tyrosine kinase ; tyrosine phosphorylation ; podosome ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D3. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115-to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115-130 kD was identified as focal adhesion kinase (p125FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p 125FAK antibody revealed that p125FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125FAK but also changed the intracellular localization of p125FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125FAK is critical for regulating osteoclast function.
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    Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 436-444 
    Details
    ISSN: 0730-2312
    Keywords: Key words ; melatonin ; glutathione ; lipopolysaccharide ; oxidative damage ; oxygen free radicals ; antioxidant ; phenobarbital ; cytochrome P450 reductase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with Nw-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals. In brain, melatonin and L-NAME increased both tGSH levels and the activity of GSH-PX in LPS-treated animals. The results suggest that melatonin protects against LPS-induced oxidative toxicity in PB-treated animals in both liver and brain, and the findings are consistent with previously published observations related to the antioxidant activity of the pineal hormone.
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    Overexpression of cellular activity and protein level of protein kinase FA/GSK-3α correlates with human thyroid tumor cell dedifferentiation (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 474-480 
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    ISSN: 0730-2312
    Keywords: kinase FA/GSK-3α ; overexpression ; thyroid ; tumor cell dedifferentiation ; carcinogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) (a member of the PDPK family) has been optimized for human thyroid tissue and used to demonstrate for the first time significantly increased (P 〈 0.001) activity in thyroid carcinoma (24.2 ± 2.8 units/mg of protein) (n = 7), thyroid adenoma (14.5 ± 2.2 units/mg of protein) (n = 6), and thyroid hyperplasia (8.0 ± 2.4 units/mg of protein) (n = 5) when compared to five normal controls (4.1 ± 1.8 units/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3α in thyroid tumor cells is due to overexpression of protein level and cellular activity of kinase FA/GSK-3α is involved in human thyroid tumor cell dedifferentiation, supporting an association of PDPK with neoplastic transformation and tumorigenesis. Since kinase FA/GSK-3α may function as a possible regulator of transcription factors/protooncogenes, kinase FA/GSK-3α may therefore play an important role in thyroid cell carcinogenesis, especially in its differentiation.
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    Growth and maturation of primary-cultured adipocytes from lean and ob/ob mice (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 455-463 
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    ISSN: 0730-2312
    Keywords: preadipocyte ; adipocyte conversion ; lipids ; triglycerides ; proliferation ; ob/ob ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Stromal vascular cells from epididymal fat pads of lean and obese mice were cultured in a medium (α-MEM) containing fetal bovine serum (FBS) and cell replication followed for 11 days. In both types of cells, confluence occurred at 4-5 days, after which virtual growth arrest occurred in lean-mouse cells while replication continued, albeit at a slower rate in obese-mouse cells. Little or no lipid accumulation or glycerol-3-phosphate dehydrogenase (GPDH) activity was observed under these conditions. When a differentiation mixture consisting of insulin, corticosterone and isobutylmethylxanthine was added to the serum-containing α-MEM, a proportion of the lean-mouse cells accumulated triglycerides and GPDH activity increased significantly, indicating differentiation. By contrast, little or no differentiation occurred in obese-mouse cells. When cells grown in serum-containing α-MEM were transferred to a serum-free defined medium at confluence, extensive differentiation and maturation occurred in lean-mouse cells but not in obese-mouse cells. Similar experiments were conducted in cells isolated from the retroperitoneal fat pad. Although the growth pattern was similar to that of epididymal preadipocytes, the retroperitoneal lean- and obese-mouse cells differentiated more readily than epididymal cells, as shown by the GPDH specific activity. These data suggest that cells from obese mice are resistant to differentiation under conditions that support extensive differentiation in lean-mouse cells.
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    U2-SnRNP B″ protein gene is an early growth-inducible gene (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 490-498 
    Details
    ISSN: 0730-2312
    Keywords: serum-stimulation ; transformation ; chromatin structure mRNA ; RNA polymerase II ; μg-small nuclear RNP ; NIH-3T3 cells ; ras oncogene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this work we isolated mouse U2-snRNP-specific b″ clones and analysed the expression of the mouse U2-snRNP-specific b″ and U1-snRNP-specific 70K genes in NIH-3T3 fibroblasts. Stimulation of growth-arrested NIH-3T3 cells with serum was found to evoke a rapid increase in the amount of cytoplasmic b″ and 70K mRNAs. These increases in mRNA did not require de novo protein synthesis. Moreover, the inhibition of protein synthesis by cycloheximide caused a superinduction in the amounts of the U1-snRNP-specific 70K transcripts. We also found that c-Ha-rasval12 oncogene-transformed NIH-3T3 cells have higher of the b″ and 70K mRNAs than the normal 3T3 cells. These data imply that the b″ and 70K are early growth response genes, and their enhanced expression might be of significance in the processing of pre-mRNAs into mature mRNAs.
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    Temporal and spatial appearance of α-dystroglycan in differentiated mouse myoblasts in culture (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 527-534 
    Details
    ISSN: 0730-2312
    Keywords: α-dystroglycan ; laminin ; myoblasts ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The dystrophin-glycoprotein complex plays an important role in muscle function. One of the components of the complex, a 156-kDa cell surface glycoprotein (α-dystroglycan) binds to laminin, thereby connecting the basal lamina and muscle cells. We have examined the progressive appearance of α-dystroglycan and laminin in muscle cells that differentiate in culture. We find that nondifferentiated cultures of C2C12 myoblasts express low amounts of dystroglycan mRNA and, in contrast, this gene is prominently expressed in differentiated myotubes. Immunofluorescence analysis with a monoclonal antibody against α-dystroglycan shows its progressive appearance during myoblast differentiation into myotubes. Immunostaining with a monoclonal antibody against laminin shows that it is not present on the surface of undifferentiated myoblasts. Subsequently, laminin becomes apparent on the surface of differentiated myotubes where it codistributes with immunostained α-dystroglycan identifies a broad band of about 140-160 kDa, resembling α-dystroglycan from rabbit muscle. The composite results indicate that α-dystroglycan and laminin appear and become co-distributed on the surface of cultured C2C12 during the progression of differentiation.
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    Nuclear ultrastructures associated with the RNA synthesis and processing (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 11-26 
    Details
    ISSN: 0730-2312
    Keywords: electron microscopy ; nuclear ultrastructures ; RNA synthesis and processing ; RNA ; mRNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Relatively a little is known about the spatial organization of RNA synthesis, processing, and transport in (mammalian) cell nuclei. This review summarizes results of electron microscopic mapping of RNA synthetic sites and macromolecules involved directly, or indirectly, in the metabolism of RNAs in somatic cell mammalian nuclei. Significance of these results will be discussed in the context of the molecular mechanisms underlying spatial arrangements of RNA metabolism. © 1995 Wiley-Liss, Inc.
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    Possible role for protein kinase C in the pathogenesis of inborn errors of metabolism (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 27-32 
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    ISSN: 0730-2312
    Keywords: Protein Kinase C (PKC) ; inborn errors of metabolism ; sphingolipidoses ; fatty acid oxidation ; signal transduction ; protein phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Protein kinase C (PKC) is a ubiquitous enzyme family implicated in the regulation of a large number of short- and long-term intracellular processes. It is hypothesized that modulation of PKC activity may represent, at least in part, a functional link between mutations (genotype) that lead to the pathological accumulation of naturally occurring compounds that affect PKC activity and perturbation of PKC-mediated substrate phosphorylation and cellular function in the corresponding diseases (phenotype). This model provides a unifying putative mechanism by which the phenotypic expression of some inborn errors of metabolism may be explained.Recent studies in a cell-free system of human skin fibroblasts support the hypothesis that alteration of PKC activity may represent the functional link between accumulation of sphingolipids and fatty acyl-CoA esters, and perturbation of cell function in sphingolipidoses and fatty acid oxidation defects, respectively. Further studies will elucidate the effects of these alterations on PKC-mediated short- and long-term cellular functions in these diseases, as well as the possible role of PKC in the pathogensis of other diseases. © 1995 Wiley-Liss, Inc.
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    Inducible expression of cyclin D1 in T-47D human breast cancer cells is sufficient for Cdk2 activation and pRB hyperphosphorylation (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 363-378 
    Details
    ISSN: 0730-2312
    Keywords: cyclin D1 function ; CDK activity ; pRB phosphorylation ; G1 phase ; cell cycle control ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The sequential transcriptional activation of cyclins, the regulatory subunits of cell cycle specific kinases, regulates progress through the cell cycle. In mitogen-stimulated cells cyclin D1 induction in early G1 is followed by induction of cyclin E, activation of the cyclin-dependent kinase Cdk2, and hyperphosphorylation of the retinoblastoma gene product (pRB) in mid-to-late G1 phase. T-47D breast cancer cells expressing cyclin D1 under the control of a metal-responsive metallothionein promoter were used to determine whether Cdk2 activation and pRB hyperphosphorylation are consequences of cyclin D1 induction. A 4-5-fold increase in cyclin D1 protein abundance was followed by approximately 2-fold increases in cyclin E protein abundance and Cdk2 activity and by hyperphosphorylation of pRB. These responses were apparent ∼ 3 h after the increase in cyclin D1 protein, and ∼ 3 h prior to the entry of cyclin D1-stimulated cells into S phase 12 h after zinc treatment. Cyclin D1 immunoprecipitates contained Cdk4 but no detectable Cdk2 and displayed pRb but not histone H1 kinase activity. Cdk2 activation was therefore likely to be due to increased abundance of cyclin E/Cdk2 complexes rather than formation of active cyclin D1/Cdk2 complexes. The sequence of events following zinc induction of cyclin D1 thus mimicked that following mitogen induction of cyclin D1. These data show that cyclin D1 induction is sufficient for Cdk2 activation and pRB hyperphosphorylation in T-47D human breast cancer cells, providing evidence that cyclin D1 induction is a critical event in G1 phase progression. © 1996 Wiley-Liss, Inc.
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    Synthesis of stromal glycosaminoglycans in response to injury (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 57-68 
    Details
    ISSN: 0730-2312
    Keywords: GAGs ; TGF-β ; stroma ; remodeling ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14,42, and 84 days. The tissue and/or discs were removed and labeled with 35S-sulfate for 18 h; GAGs were extracted with 4 M guanidine-HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS-PAGE. The nonbound fractions from the chromatography were assayed for TGF-β using Western blot analysis and for hyaluronic acid using an 125I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-β that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-β. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling. © 1995 Wiley-Liss, Inc.
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    Enhanced expression of heregulin in c-erb B-2 and c-Ha-ras transformed mouse and human mammary epithelial cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 437-446 
    Details
    ISSN: 0730-2312
    Keywords: heregulin ; transformation ; erb B-2 ; c-Ha-ras ; mammary cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Heregulin β1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor α, or amphiregulin, heregulin β1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-dependent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin β1, whereas heregulin β1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.
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    Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 484-494 
    Details
    ISSN: 0730-2312
    Keywords: ecto-enzyme ; ALP inhibitor ; Ca incorporation ; glycosylphosphatidylinositol-anchored proteins ; PI-PLC ; bone differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6-8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (Ki = 45 μM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of 45Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to 45Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells. © 1996 Wiley-Liss, Inc.
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    Alternative splicing of smooth muscle myosin heavy chains and its functional consequences (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 521-528 
    Details
    ISSN: 0730-2312
    Keywords: myosin heavy chains ; smooth muscle ; alternative splicing ; contractility ; myosin light chains ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5′ (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5′-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5′-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 ± 0.012 and 0.114 ± 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca2+/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5′-inserted MHC is not necessarily associated with contractile properties of smooth muscle. © 1996 Wiley-Liss, Inc.
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    Association of protein kinase FA/GSK-3α (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 143-150 
    Details
    ISSN: 0730-2312
    Keywords: cervical carcinoma progression ; kinase FA/GSK-3α ; overexpression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P 〈 0.001) activity in poorly differentiated cervical carcinoma (82.8 ± 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 ± 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 ± 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 ± 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3α in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3α may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3α may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3α may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression. © 1995 Wiley-Liss, Inc.
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    Tamoxifen induces TGF-β1 activity and apoptosis of human MCF-7 breast cancer cells in vitro (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 9-17 
    Details
    ISSN: 0730-2312
    Keywords: antiestrogen ; human breast cancer ; programmed cell death ; tamoxifen ; TGF-β1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.
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    Ligation of the α2-macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 39-47 
    Details
    ISSN: 0730-2312
    Keywords: α2M ; PAF ; RBF ; PKC ; lyso-PAF acetyltransferase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The binding of receptor-recognized forms of α2-macroglobulin (α2M) to macrophage α2M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca2+ mobilization. In this report, we demonstrate that ligation of the macrophage α2M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α2M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α2M+, stimulated macrophage synthesis of PAF from [3H]acetate, [3H]methylcholine, and 1-O-[3H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α2M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [3H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65-70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α2M-methylamine. By contrast, when [3H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α2M-methylamine. Both α2M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α2M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α2M to regulate levels of PAF suggests that α2M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α2M-proteinase complexes. © 1996 Wiley-Liss, Inc.
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    Decreased heterogeneity of CS histone variants after hydrolysis of the ADP-ribose moiety (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 109-117 
    Details
    ISSN: 0730-2312
    Keywords: aggregin ; chemical modification ; ADP-induced platelet responses ; NBD-Cl ; cAMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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    Effect of hypoxia on hepatic DNA methylation and tRNA methyltransferase in rat: Similarities to effects of methyl-deficient diets (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 72-80 
    Details
    ISSN: 0730-2312
    Keywords: hypoxia ; S-adenosylmethionine ; DNA methylation ; hypomethylation ; t-RNA methyltransferase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Young rats were maintained in a 10% oxygen atmosphere for 2, 6, and 10 days and administered normal rat chow and water ad libitum. Thereafter, their hepatic S-adenosyl-L-methionine (AdoMet) and activity and mRNA levels of AdoMet synthetase were assayed. AdoMet levels decreased by 45% after 10 days; hepatic AdoMet synthetase also declined by ∼40%. In rats with low hepatic AdoMet, the mRNA level of AdoMet synthetase also declined by up to 80%. No significant change in AdoMet or AdoMet synthetase was noted in pair-fed normoxic rats. DNA hypomethylation was determined in terms of incorporation of [3H]methyl of AdoMet incorporated at unmethylated sites in DNA in reactions mediated by methylases Hpall and Sssl. As compared to the normal hepatic DNA, [3H]methyl group incorporation in the 10-day hypoxic DNA was almost double in the Hpall-mediated reaction and ∼10-fold in the Sssl-mediated reaction. Hepatic tRNA methyltransferase activity doubled after 10 days of hypoxia. However, hypoxic rats showed no detectable mRNA transcripts for c-myc and c-fos oncogenes on Northern blot analysis. These observations show that because of subnormal activity of AdoMet synthetase, hypoxic liver is depleted of AdoMet, even when the animals are administered a complete diet. However, unlike rats on chronic lipotrope-deficient diets, hypoxic rats on a complete diet show no aberrant expression of oncogenes. © 1996 Wiley-Liss, Inc.
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    Cell density-dependent changes in the insulin action pathway: Evidence for involvement of protein-tyrosine phosphatases (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 31-38 
    Details
    ISSN: 0730-2312
    Keywords: cell density ; DNA synthesis ; Mr receptor substrates ; IRS-1 protein ; tyrosine phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In order to examine alterations in the phosphorylation state of proteins involved in insulin action that might accompany the reduced growth state of density-arrested cells, we measured the insulin-stimulated phosphorylation of the receptor and high Mr cellular substrates of the receptor kinase in rat hepatoma cells at different cell densities. As cell density increased from 2 × 105 to 3.2 × 106 per 35-mm well, the rate of DNA synthesis fell to 22% of control, while insulin-stimulated tyrosine phosphorylation of high Mr receptor substrates (“pp185”) was enhanced to 198% of control, without a change in the abundance of insulin receptor substrate (IRS)-1 protein. In anti-IRS-1 immunoprecipitates, tyrosine phosphorylation was increased by only 30%, suggesting that increased tyrosine phosphorylation of additional high Mr proteins (e.g., IRS-2) accounted for much of the observed increase in tyrosine phosphorylation of the receptor substrates. In spite of increased tyrosine phosphorylation of IRS-1 and total pp185-related proteins, however, cells studied at high growth density exhibited a 25% decrease in IRS-1-associated phosphatidylinositol 3′-kinase activity and only a 39% increase in phosphatidylinositol 3′-kinase activity in antiphosphotyrosine immunoprecipitates. To explore the potential role of hepatic protein-tyrosine phosphatases (PTPases) in the hyperphosphorylation of pp185 proteins, we found by immunoblotting that at high cell density the intracellular PTPase PTP18 and the transmembrane PTPase LAR were reduced in abundance by 49% and 55%, respectively, while the abundance of the SH2-domain containing PTPase SH-PTP2 was increased by 48%. These data demonstrate that the attenuation of post-receptor signaling by insulin in hepatoma cells at increasing growth density involves changes in endogenous substrate phosphorylation which may result from alterations in specific PTPases implicated in the regulation of the insulin action pathway. © 1996 Wiley-Liss, Inc.
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    Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 48-60 
    Details
    ISSN: 0730-2312
    Keywords: nuclear pore structure ; digitonin permeabilization ; immunofluorescence ; coiled-coil proteins ; Tpr ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen λgt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double-label immunofluorescent microscopy with the anti-Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349-amino acid protein with extensive coiled-coil domains and an acidic globular C-terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from 32P-orthophosphate-labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed. © 1996 Wiley-Liss, Inc.
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    Induction of mouse β integrin expression following transfection with human α4 chain (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 127-138 
    Details
    ISSN: 0730-2312
    Keywords: β1 integrin ; β7 integrin ; α/β integrin subunit association ; VLA-4/VCAM adhesion ; integrin surface expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.
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    Masthead (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995) 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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    URL: http://dx.doi.org/10.1002/jcb.240590401
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    The Rb2/p130 gene product is a nuclear protein whose phosphorylation is cycle regulated (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 402-408 
    Details
    ISSN: 0730-2312
    Keywords: tumor suppressor gene ; retinoblastoma gene ; Rb2/p130 ; pocket protein ; nuclear phosphoprotein ; E1A oncoprotein ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The Rb2/p130 protein has been shown to have a high sequence homology with the retinoblastoma gene product (pRb), one of the most well-characterized tumor suppressor genes, and with pRb-related p107, especially in their conserved pocket domains, which display a primary role in the function of these proteins. In this study, we report on the biochemical and immunocytochemical characterization of the Rb2/p130 protein, using a polyclonal antibody developed against its “spacer” region included in the pocket domain of the whole protein. We show that pRb/p130 is a phosphoprotein located at the nuclear level and that its phosphorylation pathway can be dramatically reduced by phosphatase treatment. Moreover pRb/p130, with p107, with p107, is one of the major targets of the E1A viral oncoprotein-associated kinase activity, showing a phosphorylation pattern which is modulated during the cell cycle, reaching a peak of activation at the onset of S-phase. © 1995 Wiley-Liss, Inc.
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    Masthead (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995) 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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    URL: http://dx.doi.org/10.1002/jcb.240590301
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    TGF-β receptors are diminished after retinoid exposure in rat liver epithelial cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 230-237 
    Details
    ISSN: 0730-2312
    Keywords: retinoic acid ; retinol ; binding ; transglutaminase ; hepatic ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: When rat liver epithelial cells were exposed to retinoic acid or retinol for 24 hr, the levels of transforming growth factor-β (TGF-β) receptors were reduced in a dose-dependent way. The decrease appeared after 12 hr of incubation with the retinoids and binding levels remained low until 24 hr after the removal of the molecules. Retinoid treatment induced a fourfold enhancement of transglutaminase (TGase) activity in the cell membranes, and cystamine, an inhibitor of TGase, prevented the decrease of the receptors. Neutralization of TGF-β by a monoclonal antibody did not suppress the decrease of the binding levels, indicating that decreased TGF-β binding capacity was not due merely to the internalization of ligand-bound receptors promoted by a stimulation of TGF-β synthesis. Thus, retinoid treatment resulted in an intense disappearance of the functional receptors from the membranes that seemed to be mediated by increased TGase activity. This phenomenon can represent a strong signal attenuation for TGF-β following retinoid exposure. © 1996 Wiley-Liss, Inc.
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    Overexpression of protein kinase FA/GSK-3α (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 238-245 
    Details
    ISSN: 0730-2312
    Keywords: human hepatoma ; dedifferentiation/progression ; PDPK ; overexpression ; kinase FA/GSK-3α ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P 〈 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 ± 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 ± 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 ± 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3α is due to overexpression of the protein. Elevated kinase FA/GSK-3α expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be ∼fivefold overexpressed in well differentiated hepatoma and ∼13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3α is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3α is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression. © 1996 Wiley-Liss, Inc.
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    Phenotypic expression of marrow cells when grown on various substrata (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 246-254 
    Details
    ISSN: 0730-2312
    Keywords: marrow stromal cells ; cell morphogenesis ; attachment ; ECM ; mRNA expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen 1. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. © 1996 Wiley-Liss, Inc.
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    Integrin-dependent role of human T cell matrix metalloproteinase activity in chemotaxis through a model basement membrane (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 452-458 
    Details
    ISSN: 0730-2312
    Keywords: adhesion ; migration ; protease ; lymphocyte ; immunity ; connective tissue ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human T lymphoblastoma cells of the CD4+ 8+ Tsup-1 line, that express alpha4 and alpha5 but not alpha6 integrins of the beta1 family, and CD4+ human blood T cells bind vasoactive intestinal peptide (VIP) with high affinity, leading to increased adherence, secretion of matrix metalloproteinases (MMPs), and chemotaxis. VIP-enhanced adherence of T cells to fibronectin was inhibited significantly by neutralizing monoclonal antibodies to beta1 〉 alpha4 〉〉 alpha5, but not to alpha6. Antibodies to beta1 and alpha4 suppressed to a similarly significant extent VIP stimulation of both MMP-dependent T cell chemotaxis through fibronectin-enriched Matrigel and T cell degradation of 3H-type IV collagen in the Matrigel, without affecting VIP-evoked secretion of MMP by suspensions of T cells. The lesser inhibition of VIP-enhanced adherence of T cells to fibronectin by anti-alpha5 antibody, than antibodies to beta1 or alpha4 chains, was associated with lesser or no suppression of MMP-dependent T cell chemotaxis through Matrigel and T cell degradation of type IV collagen in the Matrigel in response to VIP. Specific beta1 integrins thus mediate interactions of stimulated T cells with basement membranes, including adherence, localized digestion by MMPs, and chemotactic passage, that promote entry of T cells into extravascular tissues. © 1996 Wiley-Liss, Inc.
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    Mammalian CAP interacts with CAP, CAP2, and actin (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 459-466 
    Details
    ISSN: 0730-2312
    Keywords: adenylyl cyclase ; BAT3 ; cytoskeleton ; RAS ; signaling ; yeast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We previously identified human CAP, a homolog of the yeast adenylyl cyclase - associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts. © 1996 Wiley-Liss, Inc.
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    Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 514-528 
    Details
    ISSN: 0730-2312
    Keywords: prothrombin ; prethrombin 2 ; fragment 1.2 ; α-thrombin ; prothrombin activation ; serine proteinase ; human vascular smooth muscle cells ; mitogenic activity ; enzymatic activity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R155-S156. Cleavage at R271-T272 generated fragment 1.2 and prethrombin 2 whilst cleavage at R284-T285 yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R320-I321 which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1-3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R271-S272 and by α-thrombin at R155-S156 and R284-T285. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30-40 kDa. © 1995 Wiley-Liss, Inc.
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    Expression of basic helix-loop-helix transcription factors in explant hematopoietic progenitors (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 478-488 
    Details
    ISSN: 0730-2312
    Keywords: basic helix-loop-helix ; interleukin-1 ; interleukin-3 ; granulocyte-macrophage colony-stimulating factor ; progenitor ; transcription factor ; c-kit ligand ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The basic helix-loop-helix (bHLH) transcription factors form heterodimers and control steps in cellular differentiation. We have studied four bHLH transcription factors, SCL, lyl-1, E12/E47, and Id-1, in individual lineage-defined progenitors and hematopoietic growth factor - dependent cell lines, evaluating mRNA expression and the effects of growth factors and cell cycle phase on this expression. Single lineage-defined progenitors selected from early murine colony starts and grown under permissive conditions were analyzed by RT-PCR. SCL and E12/E47 were expressed in the vast majority of tri-, bi-, and unilineage progenitors of erythroid, macrophage, megakaryocyte, and neutrophil lineages. Expression for E12/E47 was not seen in unilineage megakaryocyte and erythroid or bilineage neutrophil/mast cell progenitors. Lyl-1 showed a more restricted pattern of expression, although expression was seen in some bi- and unilineage progenitors. No expression was detected in erythroid, erythroid-megakaryocyte-macrophage, macrophage-neutrophil, macrophage, or megakaryocytic progenitors. Id-1, an inhibitory bHLH transcription factor, was also widely expressed in all bi- and unilineage progenitors; only the trilineage erythroid-megakaryocyte-macrophage progenitors failed to show expression. Expression of these factors within a progenitor class was generally heterogeneous, with some progenitors showing expression and some not. This was seen even when two sister cells from the same colony start were analyzed. Id-1, but not E12/E47, mRNA was increased in FDC-P1 and MO7E hematopoietic cell lines after exposure to IL-3 or GM-CSF, Id-1, E12, and lyl-1 showed marked variation at different points in cell cycle in isoleucine-synchronized FDC-P1 cells. These results suggest that SCL, lyl-1, E12/E47, and Id-1 are important in hematopoietic progenitor cell regulation, and that their expression in hematopoietic cells varies in response to cytokines and/or during transit through cell cycle. © 1996 Wiley-Liss, Inc.
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    Domains of laminin (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 493-501 
    Details
    ISSN: 0730-2312
    Keywords: basement membrane ; cell binding ; epidermolysis bullosa ; extracellular matrix ; gene knock-out ; integrin ; laminin ; muscular dystrophy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Extracellular matrix molecules are often very large and made up of several independent domains, frequently with autonomous activities. Laminin is no exception. A number of globular and rod-like domains can be identified in laminin and its isoforms by sequence analysis as well as by electron microscopy. Here we present the structure-function relations in laminins by examination of their individual domains. This approach to viewing laminin is based on recent results from several laboratories. First, some mutations in laminin genes that cause disease have affected single laminin domains, and some laminin isoforms lack particular domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. Second, laminin-like domains have now been found in a number of other proteins, and data on these proteins may be informative in terms of structure-function relationships in laminin. Finally, a large body of data has accumulated on the structure and activities of proteolytic fragments, recombinant fragments, and synthetic peptides from laminin. The proposed activities of these domains can now be confirmed and extended by in vivo experiments. © 1996 Wiley-Liss, Inc.
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    Variable effects of tyrosine kinase inhibitors on avian osteoclastic activity and reduction of bone loss in ovariectomized rats (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 629-637 
    Details
    ISSN: 0730-2312
    Keywords: bone resorption ; tyrphostins ; genistein ; herbimycin ; osteoporosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 μM and 0.1 μM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 μM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 μM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 μmol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P 〈 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity. © 1996 Wiley-Liss, Inc.
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    Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 443-452 
    Details
    ISSN: 0730-2312
    Keywords: protein synthesis ; retinoic acid ; EGF ; NGF ; differentiation ; phosphatase ; elF-4E ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation.By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity.These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.
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    Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 56-68 
    Details
    ISSN: 0730-2312
    Keywords: CoA-independent transacylase ; phospholipase D ; subcellular localization ; neutrophils ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTPγS and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes. © 1996 Wiley-Liss, Inc.
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    Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: Evidence for intracellular complexes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 240-250 
    Details
    ISSN: 0730-2312
    Keywords: FGF ; receptors ; internalization ; photoactivable cross-linker ; heparan sulfate proteoglycans ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azido-benzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37°C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Flg receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4°C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4°C and then washed, were shifted to 37°C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex. © 1996 Wiley-Liss, Inc.
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    hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 275-289 
    Details
    ISSN: 0730-2312
    Keywords: nuclear matrix ; HeLa S3 cells ; 2-D gel electrophoresis ; heterogeneous nuclear ribonucleoproteins ; B23 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA. © 1996 Wiley-Liss, Inc.
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    Transcriptional control of the heme oxygenase gene in mouse M1 cells during their TPA-induced differentiation into macrophages (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 314-324 
    Details
    ISSN: 0730-2312
    Keywords: M1 cell ; heme oxygenase ; transcription ; H2O2 ; TPA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It has long been known that heme oxygenase (HO) is a key enzyme in heme catabolism and recently it was also found to acts as an oxidative stress protein to produce carbon monoxide (CO), which has similar actions to those of nitrogen monoxide (NO). Therefore, we examined transcriptional control of the HO gene in mouse M1 (myeloleukemia) cells during their differentiation into macrophages. Since the promoter region of this gene is known to have a TPA-responsive element (TRE), its expression might be regulated by a C-kinase signal transduction pathway. Then we investigated the activation of the HO gene after treatment of M1 cells with TPA and inhibitors of C-kinase. When M1 cells were treated with TPA, they differentiated into macrophage-like cells. Upon treatment with TPA, H2O2 was produced first, the nuclear proto-oncogenes fos and jun were activated, and then the HO gene was activated. The extent of transcriptional activation of the fos, jun, and HO genes in M1 cells treated with TPA was reduced by a specific inhibitor of C-kinase and a scavenger of oxygen radicals. When M1 cells were treated with H2O2 essentially the same level of transcription of the HO gene was observed, but the extent of transcriptional activation of the fos and jun genes was about half of the treatment with TPA. Super-shift assays using the TRE of the HO gene revealed that the Fos and Jun proteins from nuclei of M1 cells treated with TPA bound to the TRE, and same assays using DNA with the NF-kB motif also revealed that the active NF-kB protein from M1 cells treated with H2O2 or TPA also bound to the corresponding motif. These results strongly suggest that the HO gene in M1 cells is activated by TPA through a production of H2O2, an oxidative activation pathway of NF-kB, and a signal-transduction pathway that involves C-kinase during the differentiation of macrophages that occurs upon treatment with TPA. © 1996 Wiley-Liss, Inc.
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    Binding of MAR-DNA elements by mutant p53: Possible implications for its oncogenic functions (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 172-180 
    Details
    ISSN: 0730-2312
    Keywords: chromatin structure ; nuclear matrix ; transcriptional activation ; replication ; recombination ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrety of the genome. This function of wild-type p53 as “guardian of the genome” is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function. © 1996 Wiley-Liss, Inc.
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    Dexamethasone alters rapidly actin polymerization dynamics in human endometrial cells: Evidence for nongenomic actions involving cAMP turnover (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 251-261 
    Details
    ISSN: 0730-2312
    Keywords: dexamethasone ; actin ; polymerization ; Ishikawa cells ; cAMP ; actinomycin D ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Glucocorticoids, in addition to their well characterized effects on the genome, may affect cell function in a manner not involving genomic pathways. The mechanisms by which the latter is achieved are not yet clear. A possible means for this action may involve the actin cytoskeleton, since the dynamic equilibrium of actin polymerization changes rapidly following exposure to several stimuli, including hormones. The aim of the present work was to find out if glucocorticoids exert rapid, nongenomic effects on actin polymerization in Ishikawa human endometrial cells, which represent a well characterized in vitro cell model expressing functional glucocorticoid receptors. Short term exposure of the cells to the synthetic glucocorticoid dexamethasone resulted in an overall decrease of the G/total-actin ratio in a time- and dose-dependent manner. Specifically, in untreated Ishikawa cells the G/total-actin ratio was 0.48 ± 0.01 (n = 26). It became 0.35 ± 0.01 (n = 13, P 〈 0.01) following exposure to 10-7 M dexamethasone for 15 min. This was induced by a significant decrease of the cellular G-actin level, without affecting the total actin content, indicating a rapid actin polymerization. This conclusion was fully confirmed by direct fluorimetry measurements, that showed a significant increase of the F-actin content by 44% (n = 6, P 〈 0.001) in cells treated with dexamethasone (10-7 M, 15 min). The rapid dexamethasone-induced alterations of the state of actin polymerization were further supported by fluorescence microscopy. The latter studies showed that the microfilaments of cells pretreated with 10-7 M dexamethasone for 15 min were more resistant to various concentrations of the antimicrofilament drug cytochalasin B, compared to untreated cells, implying microfilament stabilization. The action of dexamethasone on actin polymerization seems to be mediated via specific glucocorticoid binding sites, since the addition of the glucocorticoid antagonist RU486 completely abolished its effect. Moreover, it appears to act via non-transcriptional pathways, since actinomycin D did not block the dexamethasone-induced actin polymerization. In addition, cell treatment with 10-7 M dexamethasone for 15 min fully reversed the forskolin-, but not the 8-bromo-cAMP-induced actin depolymerization. In line with these findings, the cAMP content of Ishikawa cells was decreased by 29.2% after a 15 min treatment with 10-7 M dexamethasone (n = 4, P 〈 0.01). In conclusion, our results showed that dexamethasone induces rapid, time-, and dose-dependent changes in actin polymerization dynamics in Ishikawa cells. This action seems to be mediated via cAMP, involving probably nongenomic pathways. The above findings offer new perspectives for the understanding of the early cellular responses to glucocorticoids. © 1996 Wiley-Liss, Inc.
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    Monocyte chemoattractant protein-1 gene expression in injured pig artery coincides with early appearance of infiltrating monocyte/macrophages (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 303-313 
    Details
    ISSN: 0730-2312
    Keywords: monocyte chemoattractant protein-1 ; gene expression ; pig artery ; balloon injury ; monocyte/macrophages ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are potent chemokines which attract circulating monocytes and neutrophils respectively to inflamed tissues. JE/MCP-1 gene expression has been previously studied in rabbit aortae after endothelial denudation and the rapid appearance of this transcript was thought to precede emigration of phagocytes. We now report MCP-1 gene expression following de-endothelialization of iliac arteries in the pig, a species which can develop spontaneous atherosclerosis. Using Northern blot analysis, we demonstrated that MCP-1 mRNA was rapidly induced in pig arteries at 2 h and continued to increase to reach a maximum at 8 h before returning to low levels at 16-24 h after injury. The increase seen for MCP-1 mRNA at 8 h was also observed for IL-8 mRNA but was not apparent for growth-related gene expressions, urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor-1 (PAI-1). Since smooth muscle cells, endothelial cells, and phagocytes are all capable of expressing MCP-1, we examined pig arteries for immunostaining using a monoclonal antibody to human MCP-1 (5D3-F7). At 8 h after injury, the predominant cell type staining positive for MCP-1 was the monocyte/macrophage. Staining was also observed in occasional scattered neutrophils, but MCP-1 protein could not be detected in smooth muscle cells or on extracellular matrix within the sensitivity constraints posed by our methodology. Our results are consistent with invading monocyte/macrophages having a major input into the production of this chemokine in the arterial wall following injury. The fact that MCP-1 expression accompanied monocyte/macrophage presence in damaged artery, rather than preceding it, is suggestive that continued MCP-1 expression is required for functions other than chemoattraction. © 1996 Wiley-Liss, Inc.
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    Soluble factors secreted by activated T-lymphocytes modulate the transcription of the immunosuppressive cytokine TGF-β2 in glial cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 342-355 
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    ISSN: 0730-2312
    Keywords: GLRP ; T-lymphocyte ; immune response ; central nervous system ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Coordination of the immune response to injury or disease in the brain is postulated to involve bi-directional discourse between the immune system and the central nervous system. This cross communication involves soluble mediators, including various growth factors, cytokines, and neuropeptides. In this report, we demonstrate that the supernatant from activated T-lymphocytes is able to induce the transcription of a potent cytokine, TGF-β2 in glial cells. The activating stimulus invokes signaling mechanisms distinct from known kinase or protease pathways. Activation of TGF-β2 transcription correlates with the loss of binding activity for an 80 kDa glial labile repressor protein, GLRP, to a responsive region within the TGF-β2 promoter. Although GLRP shares some characteristics with the inducible transcription factor AP-1, it appears to be distinct from known AP-1 family members. These data along with previous observations demonstrating the potent immunosuppressive activity of TGF-β2, support a model for a feedback mechanism between the activated T-lymphocytes and astrocytes via TGF-β2 to regulate the immune response. © 1996 Wiley-Liss, Inc.
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    Developmental changes in transcription factors associated with the nuclear matrix of chicken erythrocytes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 454-466 
    Details
    ISSN: 0730-2312
    Keywords: nuclear matrix ; histone H5 ; transcription ; transcription factors ; erythroid development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix has roles in organizing nuclear DNA and in controlling transcription. Transcription factors are associated with the nuclear matrix, with the spectra of transcription factors differing from one cell type to another. In this study we identified the transcription factors and enzymes functioning in the regulation of gene expression that were associated with nuclear matrix and nonmatrix nuclear fractions in erythrocytes isolated from chick embryos at different stages of development, anemic and normal adult birds. We found that the primitive erythroid nuclear matrix had the greatest histone deacetylase activity and highest levels of several transcription factors, including GATA-1, CACCC-binding proteins, and NF1. These transcription factors have key roles in erythroid-specific gene expression. The levels of these transcription factors were lower in the nonmatrix and matrix fractions isolated from definitive erythrocytes. For primitive and definitive erythrocytes, the level of CACCC-binding proteins in the nuclear matrix fraction was greater than that of Sp1. The relative levels of these transcription factors were reversed in the nonmatrix fraction. Casein kinase II was not found in erythroid nuclear matrices. The observed erythroid lineage specific alterations in erythroid nuclear matrix transcription factor composition and abundance may be involved in erythroid-specific gene expression. © 1996 Wiley-Liss, Inc.
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    Heparin-binding domain, type 1 and type 2 repeats of thrombospondin mediate its interaction with human breast cancer cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 431-442 
    Details
    ISSN: 0730-2312
    Keywords: adhesion ; breast cancer cells ; thrombospondin ; receptors ; proteoglycans ; heparin-binding peptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains of thrombospondin that mediate its binding to specific receptors on the human breast adenocarcinoma cell line, MDA-MB-231. Two recombinant fragments from the amino-terminus (TSPN18 and TSPN28), and the fusion proteins of the type 1 and type 2 repeats of human thrombospondin, inhibited binding of radiolabeled thrombospondin to MDA-MB-231 cells in suspension by 40-60% at 50 μg/ml whereas the type 3 repeat, carboxy-terminus and unfused glutathione-S-transferase as well as the synthetic peptide Gly-Arg-Gly-Asp-Ser (500 μg/ml) had little or no effect. Herapin and various glycosaminoglycans as heparan sulfate, chondroitin sulfates A, B or C, and fucoidan inhibited thrombospondin binding to MDA-MB-231 cells by more than 60% whereas dextran sulfate had only little effect. Treatment of cells with heparitinase, chondroitinase ABC, and hyaluronidase, but not with neuraminidase, induced 30-50% inhibition of thrombospondin binding suggesting the participation of both heparan sulfate and chondroitin sulfate cell surface-associated molecules. Inhibition of proteoglycan sulfation by chlorate or inhibition of glycosaminoglycan chain formation by two β-D-xylosides also led to a substantial inhibition of thrombospondin binding. Our results indicate that several domains within the thrombospondin molecule, namely the amino-terminus, type 1 and type 2 repeats, participate in its binding to specific receptors bearing sulfated glycosaminoglycans on MDA-MB-231 cells. Biological assays have indicated that, in addition to these domains, the peptide Gly-Arg-Gly-Asp-Ser inhibited MDA-MB-231 cell attachment to thrombospondin suggesting that the last type 3 repeat of the molecule may also contribute to its cell adhesive activity. © 1996 Wiley-Liss, Inc.
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    Heat shock inhibits pre-rRNA processing at the primary site in vitro and alters the activity of some rRNA binding proteins (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 506-515 
    Details
    ISSN: 0730-2312
    Keywords: heat shock ; pre-rRNA processing ; S-100 extract ; U3 snoRNA ; 3′ processing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of heat shock on pre-rRNA processing at the primary site within external transcribed spacer region 1 (ETS1) was studied in S-100 extract derived from mouse lymphosarcoma cells. In vivo labeling with [32P]orthophosphate showed that the synthesis of the rRNA precursor and its processing to 28S and 18S rRNAs were inhibited significantly due to heat shock. The processing activity was reduced by 50% at 1 h and was completely blocked following 2-h exposure of cells at 42°C. Mixing S-100 extracts from the control and heat-treated cells did not affect the processing activity in the control extract, which proves the absence of a nuclease or other inhibitor(s) of processing in the extract from the heat-shocked cells. Heat shock did not affect interaction between pre-rRNA and U3 snoRNA, a prerequisite for the processing at the primary site, but significantly altered RNA-protein interaction. Three polypeptides of 200, 110, 52 kDa that specifically cross-link to pre-rRNA spanning the primary processing site were inactivated after heat shock. Hyperthermia did not alter 3′ end processing of SV40L pre-mRNA. © 1996 Wiley-Liss, Inc.
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    Prevention and therapy of mammary cancer by monoterpenes (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 139-144 
    Details
    ISSN: 0730-2312
    Keywords: Breast cancer ; cancer prevention ; cancer therapy ; monoterpene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Monoterpenes, found in a wide variety of plants, are a major component of plant essential oils. The unsubstituted monocyclic monoterpene limonene has been shown to prevent carcinogen-induced mammary cancer at both the initiation and the promotion/progression stages. This terpene also causes the complete regression of the majority of advanced rat mammary cancer when added to the diet. Modification of limonene by hydroxylation at various positions increases both its chemopreventive and therapeutic efficacy. For example, the naturally occurring hydroxylated limonene analog perillyl alcohol is 5-10 times more potent than limonene and has a similar therapeutic index.Several cellular, metabolic and molecular activities are associated with terpene exposure. These include induction of phase I and II hepatic detoxification enzymes, selective inhibition of protein isoprenylation, inhibition of CoQ synthesis, and induction of the mannose 6-phosphate/IGFII receptor and TGFβ.Due to the therapeutic efficacy of monoterpenes in experimental model systems, clinical evaluation of this class of compounds has begun in advanced cancer patients. A Phase I trial of limonene is in progress in the UK. Efforts in the US will target perillyl alcohol for Phase I testing. Pre-IND toxicology is currently being completed. Phase I trials are anticipated to begin in the Spring of 1995. We feel that the results of these therapeutic trials, if positive, will facilitate the development of current terpenes and more potent analogs for future chemoprevention trials.
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    Oncogene alterations in primary, recurrent, and metastatic human bone tumors (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 37-50 
    Details
    ISSN: 0730-2312
    Keywords: osteosarcoma ; chondrosarcoma ; GCT ; oncogene alterations ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the structure and the expression of various oncogenes in three of the most common human bone tumors - osteosarcoma (36 samples from 34 patients), giant cell tumor (10 patients), and chondrosarcoma (18 patients) - in an attempt to identify the genetic alterations associated with these malignancies. Alterations of RB and p53 were detected only in osteosarcomas. Alterations of c-myc, N-myc, and c-fos were detected in osteosarcomas and giant cell tumors. Ras alterations (H-ras, Ki-ras, N-ras) were rare. Chondrosarcomas did not contain any detectable genetic alterations. Our results suggest that alterations of c-myc, N-myc, and c-fos oncogenes occur in osteosarcomas, in addition to those previously described for the tumor suppressor genes RB and p53. Moreover, statistical analyses indicate that c-fos alterations occur more frequently in osteosarcoma patients with recurrent or metastatic disease. © 1996 Wiley-Liss, Inc.
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    Mercuric chloride mediates a protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase which is independent of the phosphorylation / dephosphorylation of a carboxyl terminal tyrosine (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 104-114 
    Details
    ISSN: 0730-2312
    Keywords: Src kinase ; mercuric chloride ; redox ; sulfhydryl group ; receptor polymerization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl2) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg2+ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl2 at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl2-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.
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    Is DNA topoisomerase IIβ a nucleolar protein? (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 162-173 
    Details
    ISSN: 0730-2312
    Keywords: Topo IIα ; Topo IIβ ; interphase ; mitosis ; mitogenic stimulation ; nucleoplasm ; nucleolus ; lymphocytes ; HeLa ; immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have carried out immunofluorescence labelling of two human cell types, HeLa cells and peripheral blood lymphocytes, prepared by several different fixation/permeabilization protocols using a variety of antibodies against DNA Topoisomerase II (Topo II). We have found that the distribution of Topo IIα was overall similar during interphase and mitosis to that previously reported, regardless of antibody and of sample preparation. On the other hand, the interphase distribution of Topo IIβ was quite variable, depending both on the antibody and on the method used to prepare the sample. Our interpretation of the data is that, like Topo IIα, Topo IIβ is primarily a nucleoplasmic protein, but that unlike Topo IIα, small amounts are also associated with intranucleolar chromatin. © 1996 Wiley-Liss, Inc.
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    Differential gene expression of extracellular matrix components in dilated cardiomyopathy (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 185-198 
    Details
    ISSN: 0730-2312
    Keywords: extracellular matrix ; remodeling ; collagenase ; collagen ; dilated cardiomyopathy ; congestive heart disease ; end-stage heart failure ; matrix metalloproteinase ; tissue inhibitor of metalloproteinase ; differential display mRNA analysis ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Extracellular matrix metalloproteinases (MMPs) are activated in dilated cardiomyopathic (DCM) hearts [Tyagi et al. (1996): Mol Cell Biochem 155:13-21]. To examine whether the MMP activation is occurring at the gene expression level, we performed differential display mRNA analysis on tissue from six dilated cardiomyopathy (DCM) explanted and five normal human hearts. Specifically, we identified three genes to be induced and several other genes to be repressed following DCM. Southern blot analysis of isolated cDNA using a collagenase cDNA probe indicated that one of the genes induced during DCM was interstitial collagenase (MMP-1). Northern blot analysis using MMP-1 cDNA probe indicated that MMP-1 was induced three- to fourfold in the DCM heart as compared to normal tissue. To analyze posttranslational expression of MMP and tissue inhibitor of matrix metalloproteinase (TIMP) we performed immunoblot, immunoassay, and substrate zymographic assays. TIMP-1 and MMP-1 levels were 37 ± 8 ng/mg and 9 ± 2 ng/mg in normal tissue specimens (P 〈 0.01) and 2 ± 1 ng/mg and 45 ± 11 ng/mg in DCM tissue (P 〈 0.01), respectively. Zymographic analysis demonstrated lytic bands at 66 kDa and 54 kDa in DCM tissue as compared to one band at 66 kDa in normal tissue. Incubation of zymographic gel with metal chelator (phenanthroline) abolished both bands suggesting activation of neutral MMP in DCM heart tissue. TIMP-1 was repressed approximately twentyfold in DCM hearts when compared with normal heart tissue. In situ immunolabeling of MMP-1 indicated phenotypic differences in the fibroblast cells isolated from the DCM heart as compared to normal heart. These results suggest disruption in the balance of myopathic-fibroblast cell ECM-proteinase and antiproteinase in ECM remodeling which is followed by dilated cardiomyopathy. © 1996 Wiley-Liss, Inc.
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    Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3 (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 199-206 
    Details
    ISSN: 0730-2312
    Keywords: regulation of transcription ; control of proliferation ; vitamin D3 analogues ; vitamin D3 receptor ; limited protease digestion assay ; lymphocytes ; breast cancer cells ; promoter selectivity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues. © 1996 Wiley-Liss, Inc.
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    Rapamycin inhibits aldolase A expression during human lymphocyte activation (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 239-251 
    Details
    ISSN: 0730-2312
    Keywords: lymphocyte activation ; Krebs cycle ; energy metabolism ; immunosuppressives ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Rapamycin (RAPA) strongly inhibits lymphocyte activation and proliferation, but does not affect most of the activation-related gene expression at the mRNA level. In order to understand the mechanism of action of RAPA and to gain further insights in lymphocyte signalling which is impaired by RAPA, we screened for RAPA-sensitive genes using differential hybridization. The expression of human aldolase A gene was found to be inducible during T and B cell activation, and the induction was repressed by RAPA at both the mRNA and enzymatic levels. The other two important immunosuppressants, cyclosporin A and FK506, also inhibited the mitogen-induced upregulation. However, none of these three drugs inhibited the constitutive expression. There was no fluctuation of aldolase A expression during the cell cycle, and RAPA failed to block the first cell cycle after synchronization in Jurkat cells. However, the second cycle was hampered by RAPA, and this was correlated with the inhibition of aldolase A expression during this later stage. Since aldolase A is a key enzyme in glycolysis and lymphocytes mainly depend on glycolysis for energy supply, the data from this study suggest that aldolase A might be one of the downstream targets of RAPA. The inhibition of the enzyme upregulation might deprive the cells of additional supply of energy, and prevent the cells from entering an optimal status for proliferation. © 1996 Wiley-Liss, Inc.
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    Remodeling of nuclear architecture during the cell cycle in Drosophila embryos (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 268-279 
    Details
    ISSN: 0730-2312
    Keywords: nuclear matrix ; mitosis ; Drosophila embryo ; monoclonal antibody ; spindle formation ; nucleus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Little is known about what determines the nuclear matrix or how its reorganization is regulated during mitosis. In this study we report on a monoclonal antibody, mAb2A, which identifies a novel nuclear structure in Drosophila embryos which forms a diffuse meshwork at interphase but which undergoes a striking reorganization into a spindle-like structure during pro- and metaphase. Double labelings with α-tubulin and mAb2A antibodies demonstrate that the microtubules of the mitotic apparatus co-localize with this mAb2A labeled structure during metaphase, suggesting it may serve a role in microtubule spindle assembly and/or function during nuclear division. That the mAb2A-labeled nuclear structure is essential for cell division and/or maintenance of nuclear integrity was directly demonstrated by microinjection of mAb2A into early syncytial embryos which resulted in a disintegration of nuclear morphology and perturbation of mitosis. © 1996 Wiley-Liss, Inc.
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    Protein phosphatase 2A in stretch-induced endothelial cell proliferation (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 311-319 
    Details
    ISSN: 0730-2312
    Keywords: protein phosphatase 2A ; endothelial cells ; cyclic strain ; proliferation ; okadaic acid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic strain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C. © 1996 Wiley-Liss, Inc.
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  • 98
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    Immunocytochemical demonstration of a lipid droplet-specific capsule in cultured Leydig cells of the golden hamsters (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 366-373 
    Details
    ISSN: 0730-2312
    Keywords: capsule ; lipid droplet ; Leydig cell ; monoclonal antibody ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3β-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet. © 1996 Wiley-Liss, Inc.
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  • 99
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    Localization of the fructose 1,6-bisphosphatase at the nuclear periphery (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 453-462 
    Details
    ISSN: 0730-2312
    Keywords: FBPase ; gluconeogenesis ; perinuclear association ; metabolic zonation ; immunolocalization ; subcellular fractionation ; confocal microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The localization of fructose 1,6-bisphosphatase (D-Fru-1,6-P2-1-phosphohydrolase, EC 3.1.3.11) in rat kidney and liver was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from pig kidney. The immunohistochemical analysis revealed that the bisphosphatase was preferentially localized in hepatocytes of the periportal region of the liver and was absent from the perivenous region. Fructose-1,6-bisphosphatase was also preferentially localized in the cortex of the kidney proximal tubules and was absent in the glomeruli, loops of Henle, collecting and distal tubules, and in the renal medulla. As indicated by immunocytochemistry using light microscopy and confirmed with the use of reflection confocal microscopy, the enzyme was preferentially localized in a perinuclear position in the liver and the renal cells. Subcellular fractionation studies followed by enzyme activity assays revealed that a majority of the cellular fructose-1,6-bisphosphatase activity was associated to subcellular particulate structures. Overall, the data support the concept of metabolic zonation in liver as well as in kidney, and establish the concept that the Fructose-1,6-bisphosphatase is a particulate enzyme that can not be considered a soluble enzyme in the classical sense. © 1996 Wiley-Liss, Inc.
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  • 100
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    Cloning, characterization, and expression of the transforming growth factor-β type I receptor promoter in fetal rat bone cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 478-490 
    Details
    ISSN: 0730-2312
    Keywords: transcription initiation ; CpG island ; transcription factor AP2 ; transcription factor Sp1 ; osteoblasts ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transforming growth factor (TGF-β) binds several discrete membrane proteins. Of these, a type I receptor appears indispensable for signal transduction. Previous examination of TGF-β receptor expression has been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-β function and receptor expression during osteogenesis, we have now cloned a 4 kilobase (kb) DNA fragment 5' proximal to the coding region of the rat TGF-β type I receptor gene. Sequence analysis revealed multiple elements compatible with transcription initiation, including a properly positioned and oriented CCAAT box, six Sp1 binding sites (three defining GC boxes), and two strong AP2 binding sites within a 0.7 kb span directly upstream of the coding region. The 3' terminal 0.3 kb span comprises a GC-enriched (77%) so-called CpG island that, like other similarly organized promoters, lacks a TATA box. Primer extension and RNase protection studies with cRNAs from this area show multiple initiation sites within 220 bp 5' proximal to the initial methionine codon. Transient transfections using nested, deleted, and inverted promoter sequences demonstrated maximal reporter expression by a 1 kb fragment encompassing all of these elements. Truncation of the 1 kb fragment from the 5' and 3' ends indicated the need for several elements for peak promoter activity. These results, and transfections in fetal rat bone and dermal cells, suggest that this promoter contains elements that specify basal and conditional expression of the TGF-β type I receptor in bone. © 1996 Wiley-Liss, Inc.
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