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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 30 (1995), S. 272-284 
    ISSN: 0886-1544
    Keywords: tensin ; cortactin ; vinculin ; chicken ; osteoclasts ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The expression and localization of tensin and cortactin were examined in osteoclast precursors in comparison with isolated osteoclasts on various substrates. Initially, the ability of hen monocytes to differentiate into osteoclasts was evaluated on plastic or glass, and compared to differentiation on bone. Specifically, monocytes were isolated from the medullary bones of egg-laying hens maintained on a Ca-deficient diet. Differentiation was monitored morphologically and by quantitation of the ability to form Howship's lacunae in bone slices or resorb radiolabeled bone particles of 20-53 m̈m diameter. These cells differentiated into tartrate resistant acid phosphatase (TRAP)-positive, bone resorbing, multinucleated syncytia in the presence of cytosine-1-β-D-arabinofuranoside in a time dependent manner (day 1-6). Differentiation into osteoclast-like cells was similar whether cultured on plastic, on glass, or on bone. When compared to GAP-DH control levels, tensin and cortctin mRNA levels increased by 7- and 10-fold, respectively, by day 6. Tensin and cortactin protein levels also increased by 6- and 15-fold, respectively, by day 6. Immunofluorescence of differentiating precursors showed that tensin localized between regions of cell to cell contact and colocalized with vinculin in podosomes of osteoclast-like cells and of real osteoclasts. Cortactin immunofluorescence was not detectable in monocytes but localized inside tensin/vinculin podosome structures after fusion into osteoclast-like cells and in freshly isolated osteoclasts. Both tensin and cortactin were associated with attachment complexes used by osteoclast-like cells and osteoclasts to resorb bone. Specifically, punctate cortactin staining was observed inside tensin staining which formed a double ring structure at the membrane/bone interface of resorbing osteoclasts. These data showed that tensin and cortactin can be used as osteoclast differentiation markers, that participate in attachment complexes used to resorb bone, and that tensin may participate in the fusion process of osteoclast precursors. © 1995 Wiley-Liss, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 21 (1983), S. 263-275 
    ISSN: 0730-2312
    Keywords: colony-stimulating factor (CSF) ; granulocyte/macrophage colonies ; hemopoiesis ; differentiation ; glycoprotein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Colony-stimulating factor (CSF), a protein required for the in vitro formation of colonies composed of granulocytes and/or macrophages, was isolated from the urine of anemic patients by using a seven-step procedure. The purified, homogeneous CSF had a specific activity of 1.9 × 108 U/absorbance unit at 280 nm (AU). This represents an overall purification of 25,330-fold and a total recovery of 3.8%. Upon iodination of the protein, the radioactivity migrated on sodium dodecyl sulfate (SDS) gel electrophoresis as a single peak with an apparent molecular weight of 46,000; reduction with mercaptoethanol caused dissociation to a single component of molecular weight 23,000. Only the dimer is active in stimulating colony formation. Urinary CSF stimulates formation of colonies comprising only macrophages in the mouse bone' marrow cell culture assay. A neutralizing antibody raised against mouse L-cell CSF did not neutralize the activity of the urinary CSF but did bind it. This may indicate that the relative positions of antibody binding sites and the active sites are different in these two glycoproteins.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 25 (1984), S. 161-182 
    ISSN: 0730-2312
    Keywords: electrophoresis ; NEPHGE ; leukemia ; differentiation ; nuclear proteins ; G-CSF ; flourescence-activated cell sorting ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In response to a differentiation factor (G-CSF) the myelomonocytic leukemia cell line (WEHI-3B(D+) differentiates to form mature macrophages and neutrophils. The effect of G-CSF on WEHI-3B(D+) differentiation was augmented by low concentrations (5 ng/ml) of actinomycin D. Quantitative binding of an antineutrophil serum was used to segregate the differentiated cells from the leukemic blast cells. Molecular markers of later myeloid differentiation were detected in myelocytes and macrophages purified from differentiating WEHI-3B(D+ ) cells. To study the initial molecular processes associated with the initiation of WEHI-3B(D+) cells to differentiation, the protein changes were analyzed using gel electrophoresis. Quantitative analysis of the fluorographs from the two-dimensional (2D) electrophorograms of the 35S-labeled proteins revealed major changes in the biosynthetic rates for 16 proteins within 5 hr: The biosynthesis of six proteins was increased and another ten proteins were synthesized at a reduced rate. Two of the proteins (17K and 36K daltons) were located in the nucleus. Pulse-chase experiments indicated that protein turnover for these proteins was rapid but the degradation of four proteins was suppressed. At least six of the proteins (16K to 120K daltons) were acidic and were associated with the cytoplasm. Electrophoretic analysis of the 35S-labeled proteins indicated that a 35K protein induced by G-CSF was found in high abundance only in purified cells of intermediate differentiation (eg, myelocytes). Other proteins (eg, a very high molecular weight protein, and a 16K dalton protein) were obviously late markers of differentiated neutrophils or macrophages.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 37 (1988), S. 371-383 
    ISSN: 0730-2312
    Keywords: growth hormone ; adipocytes ; lipid synthesis ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It is well established that somatotropin (GH) antagonizes insulin action in vivo and that supraphysiologic concentrations of GH frequently result in insulin resistance and glucose intolerance. However, the demonstration of an anti-insulin activity by GH in vitro has been difficult. This study, therefore, set out to determine whether cultures of 3T3-L1 adipocytes could be used to examine the anti-insulin activity of GH. The ability of insulin to stimulate glucose utilization by 3T3-L1 adipocytes increases approximately five-fold during the first 4 days following treatment of the cells with a differentiation medium. It was found that glucose utilization in 3T3-L1 adipocytes is regulated in a reciprocal fashion by insulin and GH. Bovine or human GH directly inhibit up to 50% of insulin-stimulated [14C]-glucose incorporation into lipids in a concentration-dependent manner. The 3T3-L1 sensitivity to GH appears to be at the maximum (50% inhibition of an insulin response) immediately following removal of the cells from the differentiation medium and remains essentially constant during the subsequent 4 days. The GH inhibition of insulin action does not appear to be due GH enhancement of cellular degradation of insulin, competitive binding of GH to the insulin receptor, or GH-induced decrease in cell number. The 3T3-L1 adipocyte system appears to be a sensitive and reliable in vitro model with which to study the molecular mechanisms involved in both GH antagonism of insulin action and development of hormone responsiveness during cellular differentiation into adipocytes.
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  • 5
    ISSN: 0730-2312
    Keywords: differentiation ; insulin ; glucocorticoids ; glycerophosphate dehydrogenase ; adipsin mRNA's ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In the present study, we have investigated dexamethasone and insulin regulation of the expression of adipose-specific mRNA, namely, glycerophosphate dehydrogenase (G3PDH) and adipsin, at different stages of differentiation. During adipose conversion, insulin promotes an accumulation of G3PDH mRNA which is linked to cell differentiation; in fully differentiated cells, insulin is not required to maintain G3PDH gene expression. Differentiating cells in serum deprived medium already exhibit, at day I, a maximal amount of mRNA encoding for adipsin, which is tenfold decreased by 10 nM of insulin; insulin also exerts a negative effect on the abundance of adipsin mRNA in mature cells. This result indicates that adipsin appears to be a very early marker of adipose conversion, the gene expression of which is down-regulated by the presence of insulin. Dexamethasone (DEX) decreases the G3PDH message at all stages of adipose conversion, while it promotes the accumulation of adipsin mRNA mainly in differentiating cells. In DEX-treated adipocytes, the transcription efficiency of the G3PDH gene is not altered, and reduction to 50% of the message is due essentially to an approximately twofold decrease in its half-life.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 43 (1990), S. 103-110 
    ISSN: 0730-2312
    Keywords: gene expression ; differentiation ; adipose cell ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Lipoprotein lipase (LPL) and pOb24 mRNAs are known to be early markers of adipose cell differentiation. Comparative studies of the expression of pOb24 and LPL genes during adipose conversion of Ob1771 preadipocyte cells and in mouse adipose tissue have shown the following: (1) the expression of both genes takes place at confluence; this event can also be triggered by growth arrest of exponentially growing cells at the G1/S stage of the cell cycle; (2) In contrast to glycerol-3°phosphate dehydrogenase mRNA, the emergence of pOb24 and lipoprotein lipase mRNAs requires neither growth hormone or tri-iodothyronine as obligatory hormones nor insulin as a modulating hormone; (3) in mouse adipose tissue, pOb24 mRNA is present at a high level in stromal-vascular cells and at a low level in mature adipocytes, and in contrast LPL mRNAs are preferentially expressed in mature adipocytes. Thus, these two genes do not appear to be regulated in a similar manner, as also shown by the differential inhibition of their expression by tumor necrosis factor (TNF) and transforming growth factor-β (TGF-β ).
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  • 7
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 27 (1985), S. 57-65 
    ISSN: 0730-2312
    Keywords: erythropoietin ; bone marrow ; immunofluorescence ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1-2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 37 (1988), S. 21-36 
    ISSN: 0730-2312
    Keywords: leukemia ; hematopoiesis ; lectins ; western transfer ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A high-resolution technique has been used to study differentiation-related and leukemia-associated glycoproteins. Cells are labeled with the membrane-impermeable probe sulfo-N-hydroxysuccinimidyl-biotin. Nonionic detergent extracts are subjected to affinity chromatography on a number of immobilized lectins and after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) and western transfer, the biotin-labeled glycoproteins are visualized by using avidin-horseradish peroxidase and 4-chloronaphthol. With the aid of the lectins concanavalin A, Dolichos biflours agglutinin, Lens culinaris hemagglutinin, peanut agglutinin, pokeweed mitogen, Ricinus communus agglutinin I, soybean agglutinin, Ulex europeus agglutinin I (UEA), and wheat germ agglutinin, each purifies different glycoprotein subsets from the same cell type. Mature cells of distinct hematopoietic lineages differ considerably in their cell surface glycoprotein patterns.This technique was used to analyze the glycoproteins of human leukemia cells before and after the induction of differentiation. K562 cells differentiated along different lineages after treatment with phorbol 12-myristate 13-acetate, sodium butyrate, dimethyl sulfoxide, or hemin. Limited specific alterations were observed with a number of lectins when K562 erythroleukemia cells were induced to differentiate. Among these, a number of bands were identified that were either lost or appeared after induction of differentiation with all four agents. In contrast, the glycoproteins bound by UEA were drastically diminished after induction of differentiation, and the remaining UEA-bound glycoproteins bore little resemblance to those of the cells before treatment. This high-resolution technique may be useful as a general method for the examination of cell surface glycoprotein differences. Once specific glycoprotein alterations are detected, lectin affinity chromatography and SDS-PAGE allow purification of antigens for the production of monoclonal antibodies.
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  • 9
    ISSN: 0730-2312
    Keywords: U937 cell line ; differentiation ; prostaglandins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The human undifferentiated histiocytic cell-line U937 can be induced to differentiate by incubation with 12-0-tetradecanoylphorbol-13-acetate (TPA) into macrophage-like cells. Dexamethasone reduced the prostaglandin production in TPA-differentiated U937 cells dose dependently, whereas undifferentiated U937 cells were dexamethasone insensitive. Concomitantly phospholipase A2, the enzyme liberating the prostaglandin precursor arachidonic acid, was inhibited by dexamethasone in TPA-differentiated but not in undifferentiated U937 cells. The activity of lysophosphatide acyltransferase, the key enzyme of fatty acid reacylation into phospholipids, remained unchanged both in undifferentiated and TPA-differentiated U937 cells. The data suggest that responsiveness to glucocorticoid-dependent regulation of prostanoid synthesis is acquired by cells of the monocyte-macrophage lineage late in differentiation.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 188-195 
    ISSN: 0730-2312
    Keywords: leukemia ; differentiation ; growth factors ; HL-60 ; cell growth ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Downregulation of the c-myc gene in HL-60 cells is associated with growth inhibition and induction of differentiation. Previous studies have reported that the growth inhibitors TGFβ and TNFα downregulate c-myc mRNA levels, suggesting the possibility that these agents may exert some of their phenotypic effects via c-myc downregulation. Our study demonstrates that although both growth inhibitors produce a similar decrease in c-myc protein synthesis, TNFα produces a greater growth inhibition and differentiation induction in HL-60 cells. Combined addition of anti-myc oligomer with either growth inhibitor produces no additive effect. In fact, 4 μM anti-myc oligomer produces the same growth and differentiation effects as does 10 ng/ml TGFβ1. We conclude that downregulation of c-myc expression represents a common mechanism of growth inhibition by TGFβ and TNFα, but that TNFα possesses an additional effect that is independent of c-myc expression.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 50 (1992), S. 73-82 
    ISSN: 0730-2312
    Keywords: adipocyte ; differentiation ; CCAAT/enhancer binding protein ; lipoprotein lipase ; adipsin ; macrophage colony stimulating factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocyte spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of a agonists based on morphologic and gene maker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination a time dependent increase in the steady state mRNA and enzymes activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis were accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support fuction of pre-adipocyte bone marrow stormal cell lines is heterogeneous, they share a common mechanism of adipognensis. © 1992 Wiley-Liss, Inc.
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  • 12
    ISSN: 0730-2312
    Keywords: colony forming cells ; differentiation ; IL-3 ; CSF1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The ability of murine Steel factor to promote the in vitro production of granulocyte-macrophage progenitor cells (CFU-GM) was examined in short-term liquid cultures. Bone marrow from C57BL/6J or Sl/Sld mice was placed in culture for seven days with either Steel factor alone or in the presence of IL-3. CFU-GM responsive to GM-CSF, IL-3, and CSF-1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CUF-GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was protentiated by the addition of IL-3. Production of CFU-GM by C57BL/6J or Sl/Sld marrow was comparable except for enhanced production of CSF-1 responsive progenitors by Sl/Sld marrow. A recombinant Sld protein was also shown to be equivalent to the wild-type protein in its capacity to promote CFU-GM production from normal bone marrow. © 1992 Wiley-Liss, Inc.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 52 (1993), S. 463-475 
    ISSN: 0730-2312
    Keywords: preadipocytes ; differentiation ; Gsα ; Giα ; Gβ ; actin ; stress fibers ; subcellular localization ; confocal microscopy ; antibodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: HGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn2+. Gsα (largely the long isoform), Giα2, and Gβ were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP-ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. Gsα showed vesicular cytoplasmic and nuclear staining; Giα2 colocalized with actin stress fibers and disruption of these structures altered the distribution of Giα2; β subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the Giα2 and β fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G-protein subunits, although reorganization of some of these subunits does accompany actin reorganization.
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  • 14
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 53 (1993), S. 114-121 
    ISSN: 0730-2312
    Keywords: osteoprogenitor cells ; differentiation ; alkaline phosphatase ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The ability of Levamisole to decrease mineralization in skeletal tissue is usually related to its effect on alkaline phosphatase (ALP). However, Levamisole is also suspected to diminish mineralization by an additional mechanism which is unrelated to the ALP control of apatite crystal growth. To delineate the time in differentiation during which Levamisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells were propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0.2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. This suggested the existence of a Levamisole-sensitive mechanism for mineralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Levamisole decreased Day 21 mineralization when added on Days 0, 3, 5, and 7, but not when added on Day 9. The Levamisole-induced inhibition of mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to mineralize is determined during the first week of culture. The early inhibitory effect of Levamisole on mineralization was associated with increased Day 12 ALP activity.
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  • 15
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 56 (1994), S. 283-294 
    ISSN: 0730-2312
    Keywords: differentiation ; lineage ; osteogenesis ; chondrogenesis ; bone marrow ; osteoporosis ; fracture repair ; bioactive factors ; monoclonal antibodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone formation in the embryo, and during adult fracture repair and remodeling, involves the progreny of a small number of cells called mesenchymal stem cells (MSCs). These cells continuously replicate themselves, while a portion become committed to mesenchymal cell lineages such as bone, cartilage, tendon, legament and muscle. The differentiation of these cells, within each lineage, is a complex multistep pathway involving discrete cellular trasitions much like that which occurs during hematopoiesys. Progression from one stage to the next depends on the presence of specific bioactive factors, nutrients, and other environmental cues whose exquisitely controlled contributions orchestrate the entire differentiation phgenomenon. As understanding of the cellular and molecular events of osteogenic differentiation of MSCs provides the foundation for the emergence of a new therapeutic technilogy for cell therapy. The isolation and in vitro mitotic expansion of autologous human MSCs will support the development of novel protocols for the treatment of many clinically challenging conditions. For example, local bone defects can be repaired through site-directed delivery of MSCs in an appropriate carrier vehicle. Generalized conditions, such as osteoporosis, may be treatable by systemic administration of culture-expanded autologous MSCs or through biopharmaceutical regimens based on the discovery of critical regulatory molecules in the differentiation process. With this in mind, we can begin to explore therapeutic options that have never before been available.
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  • 16
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 54 (1994), S. 231-238 
    ISSN: 0730-2312
    Keywords: bone ; osteocalcin ; alkaline phosphatase ; differentiation ; halogenated hydrocarbons ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent halogenated aromatic hydrocarbon, on the development of bone tissue-like organization in primary cultures of normal diploid calvarial-derived rat osteoblasts was examined. Initially, when placed in culture, these cells actively proliferate while expressing genes associated with biosynthesis of the bone extracellular matrix. Then, post-proliferatively, genes are expressed that render the osteoblast competent for extracellular matrix mineralization and maintenance of structural as well as functional properties of the mature bone-cell phenotype. Our results indicate that, in the presence of TCDD, proliferation of osteoblasts was not inhibited but post-confluent formation of multicellular nodules that develop bone tissue-like organization was dramatically suppressed. Consistent with TCDD-mediated abrogation of bone nodule formation, expression of alkaline phosphatase and osteocalcin was not upregulated post-proliferatively. These findings are discussed within the context of TCDD effects on estrogens and vitamin D-responsive developmental gene expression during osteoblast differentiation and, from a broader biological perspective, on steroid hormone control of differentiation.
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  • 17
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    Journal of Cellular Biochemistry 58 (1995), S. 527-534 
    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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  • 18
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    Journal of Cellular Biochemistry 59 (1995), S. 443-452 
    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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  • 19
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    Journal of Cellular Biochemistry 62 (1996), S. 172-180 
    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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  • 20
    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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  • 21
    Electronic Resource
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    Journal of Cellular Biochemistry 61 (1996), S. 18-25 
    ISSN: 0730-2312
    Keywords: osteoblasts ; calvaria ; invasion ; prostate ; PC-3 cells ; differentiation ; metastasis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone metastasis is a common event and a major cause of morbidity in prostate cancer patients. After colonization of bone, prostate cells induce an osteoblastic reaction which is not associated with marrow fibrosis (i.e., osteoblast but not fibroblast proliferation). In the present study we test the hypothesis that the tumoral prostatic cell line (PC-3) secretes factors that block the osteoblast differentiation process, resulting in an increase of the relative size of the proliferative cell pool. Our results, using fetal rat calvaria cells in culture, show that conditioned medium from PC-3 cells (PC-3 CM) stimulates osteoblast proliferation and inhibits both alkaline phosphatase (AP) activity (an early differentiation marker) and the mineralization process, measured as calcium accumulation (late differentiation marker). The inhibition of the expression of AP and mineralization depends on the presence of PC-3 CM during the proliferative phase of culture and suggests that both processes occur in a nonsimultaneous fashion. The inhibitory effect of PC-3 CM was not reverted by dexamethasone, which would indicate that prostatic-derived factors and the glucocorticoid do not share a common site of action. Measurement of the proliferative capacity of subcultures from control and treated cells demonstrates that PC-3 CM treatment induces the maintenance of the proliferative potential that characterizes undifferentiated precursor cells. © 1996 Wiley-Liss, Inc.
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  • 22
    ISSN: 0730-2312
    Keywords: osteoblasts ; proliferation ; growth control ; differential display ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fetal rat calvarial-derived osteoblasts in vitro (ROB) reinitiate a developmental program from growth to differentiation concomitant with production of a bone tissue-like organized extracellular matrix. To identify novel genes which may mediate this sequence, we isolated total RNA from three stages of the cellular differentiation process (proliferation, extracellular matrix maturation, and mineralization), for screening gene expression by the differential mRNA display technique. Of 15 differentially displayed bands that were analyzed by Northern blot analysis, one prominent 310 nucleotide band was confirmed to be proliferation-stage specific. Northern blot analysis showed a 600-650 nt transcript which was highly expressed in proliferating cells and decreased to trace levels after confluency and throughout the differentiation process. We have designated this transcript PROM-1 (for proliferating cell marker). A full length PROM-1 cDNA of 607 bp was obtained by 5′ RACE. A short open reading frame encoded a putative 37 amino acid peptide with no significant similarity to known sequences. Expression of PROM-1 in the ROS 17/2.8 osteosarcoma cell line was several fold greater than in normal diploid cells and was not downregulated when ROS 17/2.8 cells reached confluency. The relationship of PROM-1 expression to cell growth was also observed in diploid fetal rat lung fibroblasts. Hydroxyurea treatment of proliferating osteoblasts blocked PROM-1 expression; however, its expression was not cell cycle regulated. Upregulation of PROM-1 in response to TGF-β paralleled the stimulatory effects on growth as quantitated by histone gene expression. In conclusion, PROM-1 represents a small cytoplasmic polyA containing RNA whose expression is restricted to the exponential growth period of normal diploid cells; the gene appears to be deregulated in tumor derived cell lines. J. Cell. Biochem. 64:106-116. © 1997 Wiley-Liss, Inc.
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  • 23
    ISSN: 0730-2312
    Keywords: osteoblasts ; calvaria ; bone formation ; proliferation ; differentiation ; osteogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have determined the age-related changes in the growth characteristics and expression of the osteoblast phenotype in human calvaria osteoblastic cells in relation with histologic indices of bone formation during postnatal calvaria osteogenesis. Histomorphometric analysis of normal calvaria samples obtained from 36 children, aged 3 to 18 months, showed an age-related decrease in the extent of bone surface covered with osteoblasts and newly synthesized collagen, demonstrating a progressive decline in bone formation during postnatal calvaria osteogenesis. Immunohistochemical analysis showed expression of type I collagen, bone sialoprotein, and osteonectin in the matrix and osteoblasts, with no apparent age-related change during postnatal calvaria osteogenesis. Cells isolated from human calvaria displayed characteristics of the osteoblast phenotype including alkaline phosphatase (ALP) activity, osteocalcin (OC) production, expression of bone matrix proteins, and responsiveness to calciotropic hormones. The growth of human calvaria osteoblastic cells was high at 3 months of age and decreased with age, as assessed by (3H)-thymidine incorporation into DNA. Thus, the age-related decrease in bone formation is associated with a decline in osteoblastic cell proliferation during human calvaria osteogenesis. In contrast, ALP activity and OC production increased with age in basal conditions and in response to 1,25(OH)2, vitamin D3, suggesting a reciprocal relationship between cell growth and expression of phenotypic markers during human postnatal osteogenesis. Finally, we found that human calvaria osteoblastic cells isolated from young individuals with high bone formation activity in vivo and high growth potential in vitro had the ability to form calcified nodular bone-like structures in vitro in the presence of ascorbic acid and β-glycerophosphate, providing a new model to study human osteogenesis in vitro. J. Cell. Biochem. 64:128-139. © 1997 Wiley-Liss, Inc.
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  • 24
    ISSN: 0730-2312
    Keywords: osteoblast ; differentiation ; replication ; osteoprogenitor ; bone marrow ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity, and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 ± 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime. J. Cell. Biochem. 64:278-294. © 1997 Wiley-Liss, Inc.
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  • 25
    ISSN: 0730-2312
    Keywords: osteocalcin ; osteonectin ; collagen ; TGF-β1 ; histone ; fibronectin ; alkaline phosphatase ; ribosomal protein S6 ; differentiation ; MC3T3-E1 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone formation involves several tightly regulated gene expression patterns of bone-related proteins. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone-related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4-10), bone matrix formation/maturation (days 10-16), and mineralization stages (days 16 -30). During the proliferation period (days 4-10), the expression of cell-cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10-16), type I collagen expression and biosynthesis, fibronectin, TGF-β1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF-β1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16-30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone-related proteins are temporally regulated during the MC3T3-E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone-related proteins in relation to the different stages during the osteoblast differentiation. © 1996 Wiley-Liss, Inc.
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  • 26
    ISSN: 0730-2312
    Keywords: nerve growth factor ; fibroblast growth factor ; K-252a ; staurosporine ; p140trk ; receptor ; signal transduction ; tyrosine kinase ; transfection ; overexpression ; PC12/endothelial hybrid cells ; DNA synthesis ; proliferation ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal-mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN-trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN-trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase Cγ, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates. J. Cell. Biochem. 66:229-244. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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  • 27
    ISSN: 0730-2312
    Keywords: vitamin D3 ; differentiation ; intracellular calcium ; store-dependent calcium influx ; cell cycle blocks ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Numerous vitamin D3 analogs (VDAs) can inhibit the proliferation of cells from several types of human malignancies. The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3(1,25D3), is formed by successive hydroxylations of cholecalciferol at the 25 and 1α positions. In this study we examined the effects of the absence of the 1α(OH) group, introduction of a double bond in position 16, and further modifications at the 23, 26, and 27 positions in the side chain on the potency of the VDAs. The parameters studied were the rapidity of the induction of monocytic differentiation, the cell cycle traverse, and the effects of VDAs on intracellular calcium homeostasis in HL60 cells. The results show that (1) 1,25D3 derivatives which lack the 1α(OH) group have little differentiation-inducing activity, (2) hexafluorination (6F) of the terminal methyl groups in the side chain partially restores the activity of 1α-desoxy compounds and potentiates the activity of 1α hydroxylated compounds, and (3) 25-(OH)-16,23E-diene-26,27-hexafluoro-vitamin D3 (Ro25-9887) alone among the twelve compounds tested induces differentiation with only minimal changes in the basal levels of intracellular calcium and store-dependent calcium influx in HL60 cells. Addition of 1α(OH) group to this compound increases its differentiation-inducing activity but also elevates basal calcium level. The results suggest that altered calcium homeostasis is not an obligatory component of HL60 leukemia cell differentiation, and that Ro25-9887 and related VDAs may be suitable for testing as components of anti-leukemic therapy. © 1996 Wiley-Liss, Inc.
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  • 28
    ISSN: 0730-2312
    Keywords: retinoblastoma family ; pRb ; p107 ; pRb2/p130 ; neuroblastoma ; differentiation ; B-myb ; c-myb ; E2F ; promoter ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins. J. Cell. Biochem. 67:297-303, 1997. © 1997 Wiley-Liss, Inc.
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  • 29
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    Journal of Cellular Biochemistry 68 (1998), S. 31-49 
    ISSN: 0730-2312
    Keywords: Bax ; Bcl-2 ; Bcl-X ; bone ; programmed cell death ; p53 ; c-fos ; Msx-2 ; differentiation ; IRF-1 ; IRF-2 ; collagenase gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We present evidence of cell death by apoptosis during the development of bone-like tissue formation in vitro. Fetal rat calvaria-derived osteoblasts differentiate in vitro, progressing through three stages of maturation: a proliferation period, a matrix maturation period when growth is downregulated and expression of the bone cell phenotype is induced, and a third mineralization stage marked by the expression of bone-specific genes. Here we show for the first time that cells differentiating to the mature bone cell phenotype undergo programmed cell death and express genes regulating apoptosis. Culture conditions that modify expression of the osteoblast phenotype simultaneously modify the incidence of apoptosis. Cell death by apoptosis is directly demonstrated by visualization of degraded DNA into oligonucleosomal fragments after gel electrophoresis. Bcl-XL, an inhibitor of apoptosis, and Bax, which can accelerate apoptosis, are expressed at maximal levels 24 h after initial isolation of the cells and again after day 25 in heavily mineralized bone tissue nodules. Bcl-2 is expressed in a reciprocal manner to its related gene product Bcl-XL with the highest levels observed during the early post-proliferative stages of osteoblast maturation. Expression of p53, c-fos, and the interferon regulatory factors IRF-1 and IRF-2, but not cdc2 or cdk, were also induced in mineralized bone nodules. The upregulation of Msx-2 in association with apoptosis is consistent with its in vivo expression during embryogenesis in areas that will undergo programmed cell death. We propose that cell death by apoptosis is a fundamental component of osteoblast differentiation that contributes to maintaining tissue organization. J. Cell. Biochem. 68:31-49, 1998. © 1998 Wiley-Liss, Inc.
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  • 30
    ISSN: 0730-2312
    Keywords: AML/CBF/PEBP2 ; regulatory element ; AML-3 ; osteoblasts ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The AML/CBFA family of runt homology domain (rhd) transcription factors regulates expression of mammalian genes of the hematopoietic lineage. AML1, AML2, and AML3 are the three AML genes identified to date which influence myeloid cell growth and differentiation. Recently, AML-related proteins were identified in an osteoblast-specific promoter binding complex that functionally modulates bone-restricted transcription of the osteocalcin gene. In the present study we demonstrate that in primary rat osteoblasts AML-3 is the AML family member present in the osteoblast-specific complex. Antibody specific for AML-3 completely supershifts this complex, in contrast to antibodies with specificity for AML-1 or AML-2. AML-3 is present as a single 5.4 kb transcript in bone tissues. To establish the functional involvement of AML factors in osteoblast differentiation, we pursued antisense strategies to alter expression of rhd genes. Treatment of osteoblast cultures with rhd antisense oligonucleotides significantly decreased three parameters which are linked to differentiation of normal diploid osteoblasts: the representation of alkaline phosphatase-positive cells, osteocalcin production, and the formation of mineralized nodules. Our findings indicate that AML-3 is a key transcription factor in bone cells and that the activity of rhd proteins is required for completion of osteoblast differentiation. J. Cell. Biochem. 66:1-8, 1997. © 1997 Wiley-Liss, Inc.
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  • 31
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    Journal of Cellular Biochemistry 72 (1998), S. 168-176 
    ISSN: 0730-2312
    Keywords: cadherin ; catenin ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cadherins form a family of cell-cell adhesion proteins that are critical to normal embryonic development. Expression of the various family members is regulated in a complex pattern during embryogenesis. Both reduced and inappropriate expression of cadherins have been associated with abnormal tissue formation in embryos and tumorigenesis in mature organisms. Evidence is accumulating that signals unique to individual members of the cadherin family, as well as signals common to multiple cadherins, contribute to the differentiated phenotype of various cell types. While a complete understanding of the regulation of cadherin expression of the molecular nature of intracellular signaling downstream of cadherin adhesion is essential to an understanding of embryogenesis and tumorigenesis, our knowledge in both areas is inadequate. Clearly, elucidating the factors and conditions that regulate cadherin expression and defining the signaling pathways activated by cadherins are frontiers for future research. J. Cell. Biochem. Suppls. 30/31:168-176, 1998. © 1998 Wiley-Liss, Inc.
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  • 32
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    Journal of Cellular Biochemistry 47 (1991), S. 184-196 
    ISSN: 0730-2312
    Keywords: glucocorticoid ; transcription ; mRNA stability ; histone ; differentiation ; bone development ; osteoblast ; promoter factors ; collagen ; osteosarcoma cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The influence of dexamethasone on expression of the osteocalcin gene which encodes the most abundant non-collagenous and only reported bone-specific protein was examined in ROS 17/2.8 osteosarcoma cells which express a broad spectrum of genes related to bone formation. Consistent with previous reports, quantitation of cellular osteocalcin mRNA levels by Northern blot analysis, osteocalcin gene transcription by activity of the osteocalcin gene promoter fused to a chloramphenicol acetyl-transferase (CAT) mRNA coding sequence following transfection into ROS 17/2.8 cells, and osteocalcin biosynthesis by radioimmunoassay indicate that dexamethasone in a concentration range of 10-6 to 10-9 M only modestly modifies basal levels of osteocalcin gene expression. However, dexamethasone significantly inhibits these parameters of the vitamin D-induced upregulation of osteocalcin gene expression in both proliferating and in confluent ROS 17/2.8 cells. In this study, we observed that the extent to which abrogation of the vitamin D response occurs is dependent on basal levels of osteocalcin gene expression as reflected by a complete inhibition of the vitamin D-induced upregulation in a ROS 17/2.8K subline with low basal expression and only a partial reduction of the vitamin D stimulation in a ROS 17/2.8C subline with eightfold higher levels of basal expression. This effect of glucocorticoid appears to be at the transcriptional and post-transcriptional levels as demonstrated by a parallel decline in the cellular representation of osteocalcin mRNA, osteocalcin gene promoter activity, and osteocalcin biosynthesis. The complexity of the glucocorticoid effect on vitamin D-mediated transcriptional properties of the osteocalcin gene is indicated by persistence of sequence-specific protein-DNA interactions at two principal osteocalcin gene promoter regulatory elements, the osteocalcin (CCAAT) box which modulates basal level of transcription, and the vitamin D responsive element, where vitamin D-mediated enhancement of osteocalcin gene transcription is controlled.
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  • 33
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    Journal of Cellular Biochemistry 47 (1991), S. 224-229 
    ISSN: 0730-2312
    Keywords: proliferation ; fibroblast growth factor ; extracellular matrix ; angiogenesis ; differentiation ; plasminogen activator ; plasminogen activator inhibitor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell-associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF-β. Interrelationships between these effects have shed some light on the mechanism of action of TGF-β and on its role in regulating EC function vis-a-vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF-β. In addition, TGF-β and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF-β has on bFGF-induced EC growth and possibly to the anti-angiogenic effect exerted by TGF-β under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF-β, thus further implicating TGF-β in the maintenance of the quiescent, differentiated aggreation of EC as found in vascular structures in vivo. While more information is needed to define what, if any, role TGF-β plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF-β are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF-β primes them for differentiation and/or is critical for the maintenance of a differentiated state.
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  • 34
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    Journal of Cellular Biochemistry 48 (1992), S. 61-72 
    ISSN: 0730-2312
    Keywords: differentiation ; neuronal cells ; GPI-anchored molecules ; metabolism ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Early postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol-anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI-anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes.The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI-PLC) was similar for all the GPI-anchored molecules, which could mean that cell-specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line.Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N-CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.
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  • 35
    ISSN: 0730-2312
    Keywords: carcinogenesis ; chemoprevention ; intermediate end point ; biomarkers ; differentiation ; growth factors ; lung cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The need for validate intermediate end point markers to facilitate lung cancer chemointervention research is competing. Three major classes of lung markers are relevant for this application. Since lung cancer includes four distinct hitologies, markers that map degrees of histologic differentiation are important. Many of the markers for squamous differentiation overlap with the candidates for application in the study of head and neck cancer. Production of tissue-specific cell product especially for surfactant or CEA is of interest, because the gene structure is known and many differentiation-related polymorphisms exist. This strategy would be useful for adenomatous type of tissue. A second type of marker is the broad group of differentiation markers. The carbohydrates or blood group-like antigens comprise a representative example. Carbohydrate structures are expressed in a specific sequence during fetal processes, and this sequence appears to reverse with the development of a cancer. Retrodifferentiation of specific differentiation markers is the basis of a major effort to effect earlier lung cancer detection using sputum immunocytochemistry. The final class includes markers which affects either positive or negative aspects of growths. Candidates in this area include growth factors or their receptors or genes that regulate growth. If the intermediate end point marker reflects tumor biology and is in that casual path of tumor progression, serial observation of that parameter should indicate the success of the intervention. In all three of these examples the clinical material to be analyzed could be sputum specimens bonrchial biopsies or resected lung tissue. Systematic analysis of these markers in context of intervention trials required to validate their utility. Long term clinical follow up will demonstrate the degree of concordance between biomarkers and more traditional clinical trial end points and will establish if such tools can play a role in catalyzing the rate of prevention research. © 1992 Wiley-Liss, Inc.
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  • 36
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    Journal of Cellular Biochemistry 50 (1992), S. 1-13 
    ISSN: 0730-2312
    Keywords: biomarkers ; chemoprevention ; differentiation ; gastrointestinal cell proliferation ; intermediate biomarker ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Dietary, environmental and genetic factors contribute to the etiology, pathogenesis and risk for gastrointestinal cancers. Measurements of cell proliferation and differentiation further identify abnormal cellular properties associated with increased susceptibility to gastrointestinal cancer. In precancerous esophagus, the proliferative compartment increases in size, increased ploidy and dysplasia develop, and epithelial cells express abnormal cytokeratins and ectopic tumor-associated antigens. In precancerous stomach, increased proliferative activity and metaplasia develop. Intestinal enzymes and mucins are expressed and normal gastric antigens are replaced by intestinal or embryonic antigens. In flat colonic mucosa and in colonic adenomas, expansions of the proliferative compartment occur. Gene expression is modified gene deletions occur and blood group-related antigens are modified as the cells undergo abnormal differentiation and develop into adenomas and carcinomas. Chemopreventive regimens are now being tested to determine whether they modify such intermediate biomarkers toward normal levels characteristic of lower risk for neoplasia. It is anticipated that the utilization of intermediate biomakers in chemoprevention studies may permit more novel chemopreventive regimens to be tested in human subjects than heretofore was possible. © 1992 Wiley-Liss, Inc.
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  • 37
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    Journal of Cellular Biochemistry 53 (1993), S. 192-198 
    ISSN: 0730-2312
    Keywords: Proliferation ; differentiation ; oncogene ; growth factors ; upper aerodigestive tract ; biomarkers ; premalignant ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The normal distribution of cell division in squamous mucosa is in the basal or adjacent suprabasal cell layers. Migration of cells toward the epithelial surface results in cell differentiation, most often expressed by high molecular weight keratin intermediate filaments and components of the cornified envelope, including “involucrin.” These latter expressions of terminal differentiation are common in keratinizing dysplasia and invasive squamous cell carcinomas. However, they are less common in the non-keratinizing dysplasias, which fail to express evidence of epithelial maturation. Cell proliferation occurs in or near the basal layer in normal or reactive/reversible hyperplasias. In dysplasia (both keratinizing and non-keratinizing), cell proliferation is observed at all levels of the epithelium. Concomitant with these abnormalities in proliferation and differentiation are nuclear changes characterized by large hyperchromatic nuclei. The enlarged nuclei reflect increased DNA content, as documented by flow cytometry and image analysis techniques. DNA aneuploidy represents a spectrum of genomic alterations reflecting steps toward the progression to invasive carcinoma, which for the most part, have not yet been identified.
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  • 38
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    Journal of Cellular Biochemistry 54 (1994), S. 354-364 
    ISSN: 0730-2312
    Keywords: cis-hydroxyproline ; rhodamine 123 ; mitochondria ; rat bone marrow ; dexamethasone ; osteoprogenitor cells ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mitochondrial response to the effect of a hydroxylase (PH) inhibitor was tested in marrow stromal cells during stimulation of osteoprogenitor cell (OPC) differentiation. The rationale for this was to explore pathways of regulatory interactions between procollagen synthesis and mitochondrial respiration that could be linked to the commitment of OPCs to mineralization. Stimulated OPCs exposed to the PH inhibitor cis-hydroxyproline (cis-HP), compared to the noninhibiting isomer trans-HP, for 11 days showed a dose-dependent decrease in cell proliferation, the surviving cells showed increased alkaline phosphatase activity. Trans-HP did not influence the cis-HP effect on ALP and on proliferation arrest. Short time exposures, 2-3 days, to cis-HP at different periods suggested that Days 0-3 and 3-5 were critical for the commitment to Day 21 mineralization of OPCs. On Days 0-3 cells were most sensitive to cis-HP, since on Day 11, 8 days after removal of cis-HP, they were too scarce to be counted by the staining method. However, the presence of 5.0 mM cis-HP in the cultures during Days 3-5 has induced on Day 21 close to 24-fold more mineralization/cell than controls, compared to the trans-HP effect, which was only close to 3-fold. The presence of cis-HP in the cultures on Days 0-3 has augmented the mitochondrial Day 3 retention of rhodamine 123 (Rho) in the stromal cells, relative to controls, compared to the presence of trans-HP. However, the presence of cis-HP during Days 3-5 or 3-6 resulted in lower Day 5 Rho retention, relative to controls, which was not significantly different from the retention that resulted from trans-HP. Since Rho retention is related to the final result of aerobic respiration level, these results are interpreted as a cis-HP inhibitory effect on procollagen peptidyl-proline hydroxylation, which may in turn release oxygen surpluses, to be available for mitochondrial consumption. The fall in Rho retention responses to cis-HP between Days 0-3 and 3-5 is suggesting either abrupt decrease in proline hydroxylation or poor mitochondrial sensitivity to oxygen in Day 3-5 cultures.
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  • 39
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    Journal of Cellular Biochemistry 55 (1994), S. 69-76 
    ISSN: 0730-2312
    Keywords: differentiation ; nuclear lamina ; intermediate filaments ; nuclear matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In the age of “virtual reality,” the imperfect microscopic silhouettes of cells and organelles are gradually being replaced by calligraphic computer drawings. In this context, textbooks and introductory slides often depict the cell nucleus as a smooth-shaped, featureless object. However, in reality, the nuclei of different cells possess distinct sizes and morphological features which develop in a programmed fashion as each cell differentiates. To dissect this complex morphogenetic process, we need to identify the basic elements that determine nuclear architecture and the regulatory factors involved. Recently, clues about the identity of these components have been obtained both by systematic analysis and by serendipity. This review summarizes a few recent findings and ideas that may serve as a first forum for future discussions and, I hope, for further work on this topic. © 1994 Wiley-Liss, Inc.
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  • 40
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    Journal of Cellular Biochemistry 58 (1995), S. 393-402 
    ISSN: 0730-2312
    Keywords: adipocytes ; bone morphogenetic proteins ; differentiation ; bone marrow stromal cells ; transforming growth factor β ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.
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  • 41
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    Journal of Cellular Biochemistry 65 (1997), S. 11-24 
    ISSN: 0730-2312
    Keywords: basic helix-loop-helix proteins ; E-box ; differentiation ; transcription ; transfection ; osteocalcin ; ld ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteoblasts undergo a temporal sequence of development characterized by transcriptional upregulation of osteoblast-specific genes. Basic helix-loop-helix (bHLH) transcription factors may control this developmental process through binding to E-box cis-acting elements in developmentally regulated genes. To investigate the role of bHLH proteins in MC3T3-E1 osteoblasts, which undergo a developmental sequence in vitro, we analyzed the transcriptional control of osteocalcin gene expression by stable transfection of an osteocalcin promoter-luciferase chimeric gene (p637OC-luc) and assessed the role of E-box cis-acting elements in osteocalcin promoter by DNA binding assays. We compared our findings in MC3T3-E1 osteoblasts with transient DNA transfections and DNA binding assays. We compared our findings in MC3T3-E1 osteoblasts with transient DNA transfections and DNA binding experiments in Ros 17/2.8 osteoblasts. We found that the activity of 637-OC luciferase promoter was low in undifferentiated 5-day-old cultures but increased in parallel with endogenous osteocalcin message expression in mature MC3T3-E1 osteoblasts, consistent with developmental stage-specific transcriptional upregulation of the osteocalcin gene. We identified two putative E-box elements in the proximal osteocalcin promoter, E-box 1 (CACATG) at - 102 and E-box 2 (CAGCTG) at position - 149. In gel mobility shift assays, factors present in nuclear extracts derived from differentiated osteoblast bound to oligonucleotide probes containing the E-box 1 and E-box 2 elements. Binding to the E-box 2 probe was not specific for the core CAGCTG element, whereas the CACATG site in E-box 1 oligonucleotide was required for specific binding of these nuclear factors. Stable transfection of p637OC-luc containing a mutant E1 site (p637OC-luc E1m), however, did not alter the developmental upregulation of osteocalcin promoter activity in MC3T3-E1 osteoblasts. Moreover, the E-box 1 mutation had no effect on either basal or vitamin D-stimulated activity of the osteocalcin promoter in Ros 17/2.8 osteoblasts in transient transfection experiments. These data suggest that osteoblasts contain undefined factors that bind to the E-box 1 CACATG site in the proximal osteocalcin promoter; however, this E-box element does not play a significant role in the developmental stage-specific regulation of the osteocalcin gene in MC3T3-E1 osteoblasts. J. Cell. Biochem. 65:11-24. © 1997 Wiley-Liss, Inc.
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  • 42
    ISSN: 0730-2312
    Keywords: vitamin D3 analogs ; 24-oxo metabolites ; growth inhibition ; differentiation ; apoptosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The seco-steroid hormone, 1α,25 dihydroxyvitamin D3 (1α,25(OH)2D3) binds to a specific nuclear receptor that acts as a ligand-inducible transcription factor. The resulting genomic effects include partial arrest in G0/G1 of the cell cycle and induction of differentiation; these effects have been observed in various types of cancer. Recently, we produced enzymatically the natural 24-oxo metabolites of 1α,25(OH)2D3 and two of its potent synthetic analogs (1α,25-(OH)2-16-ene-D3 and 1α,25-(OH)2-20-epi-D3) using a rat kidney perfusion system. We have found that the 24-oxo metabolites of both 1α,25(OH)2D3 and its analogs have either the same or greater antiproliferative activity against various cancer cells as their parental compounds. Notably, two cell lines (DU-145 (prostate cancer) and MDA-MB-436 [breast cancer]) that were extremely resistant to the antiproliferative effects of vitamin D3 analogs displayed greater sensitivity towards the 24-oxo metabolite of the vitamin D3 analog. Similarly, the 24-oxo metabolites had the capacity to induce differentiation and apoptosis and to diminish the proportion of cells in S phase. Most interestingly, while the analog 1α,25(OH)2-20-epi-D3 induced expression of BRCA1 in MCF-7 breast cancer cells; its 24-oxo metabolite dramatically suppressed BRAC1 expression. Thus, we have shown for the first time that the various biological activities produced by the hormone 1α,25(OH)2D3 and some of its analogs may represent a combination of actions by the hormone 1α,25(OH)2D3 and its natural 24-oxo metabolites. J. Cell. Biochem. 66:413-425, 1997. © 1997 Wiley-Liss, Inc.
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  • 43
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    Keywords: differentiation ; osteoblasts ; cyclin E-associated kinase ; cyclin dependent kinase inhibitors ; RB related proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Spontaneous differentiation of normal diploid osteoblasts in culture is accompanied by increased cyclin E associated kinase activity on (1) the retinoblastoma susceptibility protein pRB, (2) the p107 RB related protein, and (3) two endogenous cyclin E-associated substrates of 78 and 105 kD. Activity of the differentiation-related cyclin E complexes (diff.ECx) is not recovered in cdc2 or cdk2 immunoprecipitates. Phosphorylation of both the 105 kD endogenous substrate and the p107 exogenous substrate is sensitive to inhibitory activity (diff.ECx-i) present in proliferating osteoblasts. This inhibitory activity is readily recruited by the cyclin E complexes of differentiated osteoblasts but is not found in cyclin E immunoprecipitates of the proliferating cells themselves. Strong inhibitory activity on diff.ECx kinase activity is excerted by proliferating ROS 17/2.8 osteosarcoma cells. However, unlike the normal diploid cells, the diff.ECx-i activity of proliferating ROS 17/2.8 cells is recovered by cyclin E immunoprecipitation. The cyclin-dependent kinase inhibitor p21CIP1/WAF1 inhibits diff.ECx kinase activity. Thus, our results suggest the existence of a unique regulatory system, possibly involving p21CIP1/WAF1, in which inhibitory activity residing in proliferating cells is preferentially targeted towards differentiation-related cyclin E-associated kinase activity. J. Cell. Biochem. 66:141-152, 1997. © 1997 Wiley-Liss, Inc.
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  • 44
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    Journal of Cellular Biochemistry 65 (1997), S. 308-324 
    ISSN: 0730-2312
    Keywords: PO-B ; HL-60 ; differentiation ; AP-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: PO-B was originally characterized as a transcriptional regulatory factor of the pro-opiomelanocortin (POMC) gene; however, it has become increasingly clear that this protein may be active in tissues outside the pituitary, since it is present in diverse cell types, including differentiated HL-60 promyelocytic leukemia cells. We previously showed that PO-B DNA-binding is progressively induced during differentiation of promyelomonocytic leukemia HL-60 cells to the macrophage-like lineage (with phorbol esters). We now report that PO-B DNA-binding in HL-60 cells is similarly induced during differentiation to the granulocytic lineage (with either retinoic acid or dimethylsulfoxide). Either a genetic or pharmacologic blockade of HL-60 differentiation prohibited these inductive effects. These studies have prompted our interest in the dynamics of other transcription factor changes during HL-60 differentiation. Of these, we observed that another transcription factor (AP-1) is also robustly induced at the DNA-binding level during macrophagelike HL-60 differentiation, but not during granulocytic differentiation. Conversely, the DNA-binding of the transcription factor AP-2 was slightly reduced by TPA-induced HL-60 differentiation but unchanged during granulocyte differentiation. From these data, we conclude that the induction of PO-B DNA binding is a general marker of HL-60 myelomonocytic differentiation, but that qualitative aspects of the induction of additional distinct transcription factors, such as AP-L may contribute to lineage-specific determinants of cell fate. J. Cell. Biochem. 65:308-324. © 1997 Wiley-Liss, Inc.
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  • 45
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    Journal of Cellular Biochemistry 68 (1998), S. 74-82 
    ISSN: 0730-2312
    Keywords: cell culture ; nuclei ; nuclear degradation ; endonucleases ; polycytosine degradation ; differentiation ; cornification ; stratum corneum ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Desquamin is a glycoprotein that we have isolated from the upper granular layer and the stratum corneum of human epidermis; it is not ordinarily expressed in submerged cultures, whose terminal differentiation stops short of formation of these layers. The exogenous addition of desquamin to human cultured keratinocytes extended their maturation, and hematoxylin staining indicated a loss of cell nuclei. For confirmation, cultured cells were lysed in situ, and the nuclei were incubated with desquamin for several days, then stained with hematoxylin. Damage to the nuclei was evident: the nuclear inclusions remained intact, while the surrounding basophilic nuclear matrix was degraded. Desquamin was then tested directly for nuclease activity. Ribonuclease activity was determined by incubating desquamin with human epidermal total RNA and monitoring the dose-dependent disappearance of the 28S and 18S ribosomal RNA bands in an agarose/formaldehyde gel. On RNA-containing zymogels, we confirmed the RNase activity to be specific to desquamin. Using synthetic RNA homopolymers, we found the active RNase domains to be limited to cytosine residues. On the contrary, DNA was not degraded by an analogous procedure, even after strand-separation by denaturation. J. Cell. Biochem. 68:74-82, 1998. © 1998 Wiley-Liss, Inc.
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  • 46
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    Journal of Cellular Biochemistry 68 (1998), S. 436-445 
    ISSN: 0730-2312
    Keywords: mouse ; PDI family proteins ; retinoic acid ; dibutyryl cAMP ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be co-immunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation. J. Cell. Biochem. 68:436-445, 1998. © 1998 Wiley-Liss, Inc.
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  • 47
    ISSN: 0730-2312
    Keywords: analog ; bone ; growth inhibition ; differentiation ; vitamin D ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 1,25-Dihydroxyvitamin D3 (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D3 analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20-epi analog, CB1093 (20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo-1α,25(OH)2D3), on the proliferation and differentiation of human MG-63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG-63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 μM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6-h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog-treated cells than with 1,25D-treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR-VDRE binding with analog-treated MG-63 cells than with 1,25D-treated cells. The differences in the DNA binding of 1,25D-bound vs. analog-bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRβ proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRβ-complex with additional nuclear proteins. J. Cell. Biochem. 70:414-424, 1998. © 1998 Wiley-Liss, Inc.
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  • 48
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    Journal of Cellular Biochemistry 50 (1992), S. 65-71 
    ISSN: 0730-2312
    Keywords: chemoprevention ; colorectal cancer ; differentiation ; expansion of the proliferative compartment ; intermediate biomarker ; proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Intermediate biomarkers of abnormal cell growth and development have recently been used in chemoprevention trials in attempts to identify the efficacy of chemopreventive agents in human subjects. Measurements carried out include those related to cell proliferation, differentiation, and gene structure and expression in the colon. Among modified patterns of cell proliferation identified by microautoradiographic or immunoperoxidase assays, a characteristic expansion in the size of the proliferative compartment has been observed in normal-appearing colorectal mucosa of human subjects with diseases increasing cancer risk: the same patterns have been induced by chemical carcinogens in rodents. Moreover, this intermediate biomarker has been modulated by chemopreventive agents in both rodents and humans. Newer intermediate biomarkers being studied for application to human chemoprevention programs include normal and abnormal patterns of expression of mucins, intermediate filaments and cytoskeletal proteins, and the structure and expression of a variety of genes associated with normal and abnormal cell development. The application of these various intermediate biomarkers to chemoprevention studies is increasing the ability of investigators to analyze the efects of novel chemopreventive agents in the colon and in other organs. © 1992 Wiley-Liss, Inc.
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  • 49
    ISSN: 0730-2312
    Keywords: actin ; muscle cells ; differentiation ; cells contacts ; peptide mapping ; posttranslational control ; EDTA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The expression of vasuclar smooth muscle (VSM) α-actin mRNA during BC3H1 myogenic cell differentiation is specifically stimulated by conditions of high cell density. Non-proteolytic dissociation of cell-cell and cell-matrix contacts in post-confluent cultures of BC3H1 myocytes using EDTA promotes loss of the differentiated morphological phenotype. EDTA-dispersed myocytes exhibit an undifferentiated fibroblastoid appearance and contained reduced levels of both VSM and skeletal α-actin mRNA. Muscle α-actin mRNA levels in EDTA-dispersed myocytes were not restored to that observed in confluent myocyte preparations by experimental manipulation of cell density conditions. Pulse-labeling techniques using L-[35S] cysteine to identify muscle actin biosynthetic intermediates revelated that EDTA-dispersed myocytes expressed nascent forms of both the VSM and skeletal muscle α-actin polypetide chains. However EDTA-dispersed myocytes were less effieicent in the post-translational processing of immautre VSM α-actin compared to non-dispersed myocytes. Simple cell-to-cell contact may mediate VSM α-actin processing efficiency since high-density preparations of EDTA-dispersed myocytes processed more VSM α-actin intermediate than myocytes plated at low density. The actin isoform selectivity of the response to modulation of intercellular contacts suggests that actin biosynthesis in BC3H1 myogenic cells involves mehcanisms capable of discriminating between different isoform classes of nascent actin polypetide chains. © 1992 Wiley-Liss, Inc.
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  • 50
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    Journal of Cellular Biochemistry 55 (1994), S. 190-199 
    ISSN: 0730-2312
    Keywords: osteoclast ; osteocalcin ; bone marrow ; differentiation ; resorption ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either α-MEM with 2% heat-inactivated horse serum alone (α) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal α medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase-positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 μg/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 ± 14.2) than in GM-CSF alone (53.3 ± 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption. © 1994 Wiley-Liss, Inc.
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  • 51
    ISSN: 0730-2312
    Keywords: alkaline phosphatase ; Ishikawa endometrial cells ; butyrate ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The addition of 2mM sodium butyrate to monolayers enhances differentiation of Ishikawa endometrial cells. Cells from this cell line have been shown to enlarge and lift off the dish into dome structure over a period of 24-48 h in response to a factor in fetal bovine serum (FBS) [Fleming, 1995 J Cell Biochem in press]. When butyrate is added to monolayers, together with FBS, three- to fourfold higher numbers of differentiated structures, domes and predomes, can be counted. It had previously been shown [Holinka et al., 1986b] that estradiol induces heat stable placental alkaline phosphatese in lshikawa cells. The addition of butyrate, on the other hand, results in a significant increase in levels of a heat labile alkaline phosphatase isozyme. The heat labile isozyme is also increased to some extent in cells stimulated to differentiate in response to FBS in the absence of butyrate. Differential inhibition by homoarginine and phenylalanine indicates that butyrate is inducing the liver-bone kidney isozyme that is found in endometrial glands in vivo.
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  • 52
    ISSN: 0730-2312
    Keywords: osteoblast ; bone ; parathyroid hormone ; receptor ; differentiation ; collagen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and β-glycerophosphate, and samples were collected from 0-26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 125I-PTHrP. The numbers of receptors per μg DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1 cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro. © 1996 Wiley-Liss, Inc.
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  • 53
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    Journal of Cellular Biochemistry 67 (1997), S. 423-431 
    ISSN: 0730-2312
    Keywords: differentiation ; E1A-binding proteins ; DNA tumor viruses ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this review, the complex relationship between tissue-specific transcription factors and genes regulating cell cycle is taken into account. Both E1-A binding proteins belonging to the family of the retinoblastoma gene product and the CBP/p300 coactivator of transcription interact physically and functionally with tissue-specific transcription factor. The relationship between these two classes of molecules regulates cell fate in differentiating cells, deciding whether cells continue to replicate, undergo apoptosis or terminally differentiate. We provide here an update on the recent advances in this field and some models of interaction between E1A binding protein and tissue-specific transcription factors. J. Cell. Biochem. 67:423-431, 1997. © 1997 Wiley-Liss, Inc.
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  • 54
    ISSN: 0730-2312
    Keywords: vitamin D ; analogue ; chondrocytes ; nongenomic ; differentiation ; 1,25-(OH)2D3 ; 24,25-(OH)2D3 ; proteoglycan ; protein kinase C ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 1,25-(OH)2D3 and 24,25-(OH)2D3 mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)2D3 which have been modified on the A-ring and C,D-ring side chain (1α-(hydroxymethyl)-3β-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YA = 3a) and 1β-(hydroxymethyl)-3α-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)2D3 and 24,25-(OH)2D3. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)2D3. Chondrocyte proliferation ([3H]-thymidine incorporation), proteoglycan production ([35S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)2D3, 24,25-(OH)2D3, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)2D3 and 24,25-(OH)2D3. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)2D3 and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)2D3 having a greater effect. 24,25-(OH)2D3 caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)2D3, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation-dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Biochem. 66:457-470, 1997. © 1997 Wiley-Liss, Inc.
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  • 55
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    Journal of Cellular Biochemistry 67 (1997), S. 451-465 
    ISSN: 0730-2312
    Keywords: bone ; differentiation ; nuclear matrix ; osteoblast ; topoisomerase II ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The molecular mechanisms that mediate the transition from an osteoprogenitor cell to a differentiated osteoblast are unknown. We propose that topoisomerase II (topo II) enzymes, nuclear proteins that mediate DNA topology, contribute to coordinating the loss of osteoprogenitor proliferative capacity with the onset of differentiation. The isoforms topo II-α and -β, are differentially expressed in nonosseous tissues. Topo II-α expression is cell cycle-dependent and upregulated during mitogenesis. Topo II-β is expressed throughout the cell cycle and upregulated when cells have plateaued in growth. To determine whether topo II-α and -β are expressed in normal bone, we analyzed rat lumbar vertebrae using immunohistochemical staining. In the tissue sections, topo II-α was expressed in the marrow cavity of the primary spongiosa. Mature osteoblasts along the trabecular surfaces did not express topo II-α, but were immunopositive for topo II-β, as were cells of the marrow cavity. Confocal laser scanning microscopy was used to determine the nuclear distribution of topo II in rat osteoblasts isolated from the metaphyseal distal femur and the rat osteosarcoma cells, ROS 17/2.8. Topo II-α exhibited a punctate nuclear distribution in the bone cells. Topo II-β was dispersed throughout the interior of the nucleus but concentrated at the nuclear envelope. Serum starvation of the cells attenuated topo II-α expression but did not modulate expression of the β-isoform. These results indicate that the loss of osteogenic proliferation correlates with the downregulation of topo II-α expression. J. Cell. Biochem. 67:451-465, 1997. © 1997 Wiley-Liss, Inc.
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  • 56
    ISSN: 0730-2312
    Keywords: AML/CBF/PEBP2 ; CBFa1 ; differentiation ; osteoblasts ; regulatory elements ; transforming growth factor-β ; receptor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Organization of the transforming growth factor-β (TGF-β) type I receptor (TRI) promoter predicts constitutive transcription, although its activity increases with differentiation status in cultured osteoblasts. Several sequences in the rat TRI promoter comprise cis-acting elements for CBFa (AML/PEBP2α) transcription factors. By gel mobility shift and immunological analyses, a principal osteoblast-derived nuclear factor that binds to these sites is CBFa1(AML-3/PEBP2αA). Rat CBFa1 levels parallel expression of the osteoblast phenotype and increase under conditions that promote mineralized bone nodule formation in vitro. Fusion of CBFa binding sequence from the TRI promoter to enhancer-free transfection vector increases reporter gene expression in cells that possess abundant CBFa1, and overexpression of CBFa increase the activity of transfected native TRI promoter/reporter plasmid. Consequently, phenotype-restricted use of cis-acting elements for CBFa transcription factors can contribute to the high levels of TRI that parallel osteoblast differentiation and to the potent effects of TGF-β on osteoblast function. J. Cell. Biochem. 69:353-363. © 1998 Wiley-Liss, Inc.
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  • 57
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    Journal of Cellular Biochemistry 71 (1998), S. 467-478 
    ISSN: 0730-2312
    Keywords: cell cycle ; kinase ; signal transduction ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice. J. Cell. Biochem. 71:467-478, 1998. © 1998 Wiley-Liss, Inc.
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  • 58
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    Journal of Cellular Biochemistry 71 (1998), S. 96-108 
    ISSN: 0730-2312
    Keywords: androgens ; androgen receptor ; antiandrogens ; differentiation ; osteoblasts ; proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (∼4,000/nucleus) of androgen receptors (AR). Treatment with 5α-dihydrotestosterone (5α-DHT) inhibited the proliferation of hFOB/AR-6 cells in a dose-dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (〈200/nucleus). In hFOB/AR-6 cells, co-treatment with the specific AR antagonist, hydroxyflutamide abolished 5α-DHT-induced growth inhibition. Steady-state levels of transforming growth factor-β1 (TGF-β1) and TGF-β-induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR-6 cells with 5α-DHT, suggesting a role for the TGF-β1-TIEG pathway in mediating 5α-DHT-induced growth inhibition of hFOB/AR-6 cells. In support of this, co-treatment of hFOB/AR-6 cells with TGF-β1 (40 pg/ml) reversed the 5α-DHT-induced growth inhibition, whereas TGF-β1 alone at this dose had no effect on hFOB/AR-6 cell proliferation. Furthermore, treatment of hFOB/AR-6 cells with 5α-DHT and testosterone (10-8 M) inhibited basal and 1,25-(OH)2D3-induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast. J. Cell. Biochem. 71:96-108, 1998. © 1998 Wiley-Liss, Inc.
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  • 59
    ISSN: 0730-2312
    Keywords: cell cycle control ; histone gene expression ; S-phase ; regulatory signals ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Histone gene expression is restricted to the S-phase of the cell cycle. Control is at multiple levels and is mediated by the integration of regulatory signals in response to cell cycle progression and the onset of differentiation. The H4 gene promoter is organized into a series of independent and overlapping regulatory elements which exhibit selective, phosphorylation-dependent interactions with multiple transactivation factors. The three-dimensional organization of the promoter and, in particular, its chromatin structure, nucleosome organization, and interactions with the nuclear matrix may contribute to interrelationships of activities at multiple promoter elements. Molecular mechanisms are discussed that may participate in the coordinate expression of S-phase-specific core and H1 histone genes, together with other genes functionally coupled with DNA replication.
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  • 60
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    Journal of Cellular Biochemistry 57 (1995), S. 62-70 
    ISSN: 0730-2312
    Keywords: c-fos ; calvaria ; femur ; osteogenesis ; proliferation ; differentiation ; osteoblast ; mRNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To delineate the implication of c-fos protooncogenic in the osteogenie process, we have investigated the temporal pattern of c-fos mRNA expression in fetal and neonatal rat bone during intramembranous and endochondral bone formation. Northern blot analysis of mRNA extracted from calvaria and femur showed that expression of c-fos, Histone H4, and osteocalcin mRNAs followed a temporal sequence during bone development. The levels of histone H4 mRNA, a marker of cell proliferation, were high at early stages of fetal development of calvaria and femur, and decreased until birth. In both the postnatal calvaria and femur, c-fos mRNA levels increased transiently at birth and preceded a rise in osteocalcin transcripts, a marker of the mature osteoblast phenotype. The immunohistochemical analysis showed that c-fos protein was expressed in osteoprogenitor cells in the perichondrium and periosteum, and not in mature osteoblasts which expressed markers of differentiated osteoblasts such as type-I collagen, bone sialoprotein, and osteocalcin. Thus, the transient c-fos proto-oncogene expression during the postnatal life that precedes the osteocalcin expression may be involved in the transition from the precursor state to mature osteoblasts. These results suggest that c-fos proto-oncogene may play an important role in osteogenesis during rat postnatal life.
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  • 61
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    Journal of Cellular Biochemistry 56 (1994), S. 559-567 
    ISSN: 0730-2312
    Keywords: human ; myeloid ; nuclear ; differentiation ; chronic myeloid leukemia ; Burkitt's lymphoma ; Epstein-Barr virus ; interferon-α ; PHA ; phorbol ester ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The expression of the human myeloid cell nuclear differentiation antigen (MNDA) was observed specifically in cells of the granulocyte-macrophage lineage in our earlier reports. The specificity of MNDA expression for cells in the granulocyte-macrophage lineage was reexamined in cell line established from patients with philadelphia chromosome-positive chroni myeloid leukemia. Cell lines that expressed MNDA exhibited myeloid cell features and granulocyte or monocyte defferentiation could be induced in vitro, while cell lines exhibiting properties of very early stage cells of multipotential cells ded not express MNDA. Cells orginating from cases of burkitt's lymphoma were negative. By contrast, three Iymphoblastoid cell lines (immortalized in vitro with Epstein-Barr virus) were weakly positive and MNDA was up-regulated by interferon-α (IFN-α) treatment. As we reported previously, MNDA mRNA level in adherent monocytes is elevated by IFN-α; in this study, we further assessed MNDA expression in in vitro monocyte-derived macrophages. Three addditional agents (endotoxin, phytohemagglutinin, and photbol ester) and other conditions that affect function, cutokine production, defferentiation, and/of growth of monocytes were examined for their ability to alter MNDA expression. The results varied with the agent, cell type, and stage of differentiation. Changes in MNDA expression occurred slowly (hours to days), suggesting that MNDA could mediate changes realized over a long period. The results also reveal a discordance in certain MNDA Positiva cells between steady-state levels of changes in levels of protein and mRNA indicating that the regulation of MNDA expression occurs at more than one point. Changes in MNDA expression are consistent with a role in opposing macrophage defferentiation and activation of monocytes/macrophages.
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  • 62
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    Journal of Cellular Biochemistry 57 (1995), S. 262-270 
    ISSN: 0730-2312
    Keywords: Ishikawa cells ; monolayer ; domes ; polarization ; budding ; progesterone ; differentiation ; endometrium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human epithelial cells of the Ishikawa endometrial line can be stimulated to differentiate and form multicellular structures in 4-5 day-old monolayer cultures by the addition of a protein factor from fetal bovine serum. Multicellular structures become obvious over an 18-30-h period as the cells enlarge, separate from the dish, and form domes. These structures are similar to those that result from polarization in other epithelial cell lines. Ishikawa dome formation appears to be a multistage process. The appearance of enlarged differentiated cells is detected within hours of adding fetal bovine serum; these enlarged cells lift off the surface of the dish within 6-8 more hours. Domes are observed about 24 h after the addition of fetal bovine serum. Sometimes dome cells migrate into a “bud-like” structure that extends out from the dome. Differentiation of the domes is dependent on a factor from fetal calf serum that behaves similarly to a very large protein or complex of proteins, greater than 300 kd. Progesterone appears to enhance the formation of domes but does not elicit dome formation in the absence of serum factor.
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  • 63
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    Journal of Cellular Biochemistry 61 (1996), S. 167-171 
    ISSN: 0730-2312
    Keywords: encystment ; differentiation ; amoeba ; osmolarity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Like many yeasts, bacteria, and other sporulating microorganisms, Acanthamoeba castellanii (Neff), a free-living amoeba with pathogenic relatives, differentiates into a dormant form when deprived of nutrients. Acanthamoeba cysts redifferentiate into trophozoites when food is resupplied. We report here that Acanthamoeba encystment is also triggered by elevated osmolarity, and that osmolarity and cell surface receptor binding are synergistic in triggering differentiation. Additions of sodium chloride or glucose to rich growth media were used to produce specific osmolarity increases and similar encystment results were obtained with either additive. Although many organisms, including Acanthamoeba and mammalian cells, have been shown to adapt to hyperosmolar conditions, this is the first demonstration that hyperosmolarity can be a primary differentiation signal. © 1996 Wiley-Liss, Inc.
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  • 64
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    Journal of Cellular Biochemistry 59 (1995), S. 74-91 
    ISSN: 0730-2312
    Keywords: Apoptosis ; carcinogenesis ; cell cycle ; differentiation ; growth factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ca2+ and Ca2+-binding proteins are involved in running the cell cycle. Ca2+ spikes and signals from integrin-activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca2+ spike trigger mitotic prophase. Another Ca2+ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca2+ finally stimulates cytoplasmic division (cytokinesis).However, Ca2+ does more than this in epithelial cells, such as those lining the colon, and skin keratinocytes. These cells also need Ca2+, integrin signals, and only a small amount (e.g., 0.05-0.1 mM) of external Ca2+ to start DNA replication. Signal from their surface Ca2+ receptors trigger a combination of differentiation and apoptosis (“diffpoptosis”) when external Ca2+ concentration reaches their setpoints. The skin's steep, upwardly directed, Ca2+ gradient has a low concentration in the basal layer to allow stem and precursor keratinocytes to proliferate, and higher concentrations in the suprabasal layers to trigger the differentiation-apoptosis (“diffpoptosis”) mechanism that converts granular cells into protective, hard-shelled, dead corneocytes. A similar Ca2+ gradient may exist in the colon crypt allowing the stem cell and its amplifying transit or precursor offspring to cycle in the lower parts of the crypt, while stopping proliferation and stimulating terminal differentiation in the upper crypt and flat mucosa.Raising the amount of Ca2+ in fecal water above a critical level reduces proliferation and thus colorectal carcinogenesis in normal rats and some high-risk humans. But during carcinogenesis the Ca2+ sensors malfunction or their signals become ineffective: high Ca2+ does not stop, and may even stimulate, the proliferation of initiated mutants. Therefore, Ca2+ may either not affect, or even promote, the growth of epithelial cells in carcinogen-initiated rat colon and human adenoma patients. Clearly, a much greater understanding og how Ca2+ controls the proliferation and differentiation of epithelial cells and why initiated cells lose their reponsiveness to Ca2+ are needed to asses the drawbacks and advatages of using Ca2+ as a chemopreventor.
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  • 65
    ISSN: 0730-2312
    Keywords: hepatocytes ; differentiation ; tissue sections ; Gsα ; Giα ; Gβ ; actin ; stress fibres ; subcellular localization ; immunoflourescence microscopy ; adenylyl cyclase ; antibodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via Gsα were without effect, which was consistent with the localization of Gsα in the cytoplasmic and nuclear compartments. The “relocalization” of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions. © 1996 Wiley-Liss, Inc.
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  • 66
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    Journal of Cellular Biochemistry 65 (1997), S. 259-266 
    ISSN: 0730-2312
    Keywords: differentiation ; Gsα ; Giα ; Gβ ; actin ; stress fibres ; vimentin ; subcellular localization ; immunofluorescence microscopy ; adenylyl cyclase ; antibodies ; lipid ; fat accumulation ; obese ; obesity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The subcellular localization of Gsα, Giα1&2, Giα3, and Gβ was studied in primary-cultured undifferentiated and differentiated, lipid replete, adipose cells. The results show a distinct distribution for each of these G-proteins and differences between differentiated and undifferentiated cells. All the G-proteins examined had a cytoplasmic localization; only Giα1 and 2 showed a significant colocalization with the plasma membrane and this only in differentiated cells. Most studies using cells in culture have reported an intracellular localization for G-proteins, whereas in tissue sections the localization has been reported to be largely with the plasma membrane, with some intracellular localization. The results suggest that the cell-cell interactions or the specific geometry imposed by culture conditions favor the intracellular compared to peripheral localization of G-proteins. Alternately, the posttranslational modifications necessary for G-protein insertion in the plasma membrane may be deficient in cultured cells. J. Cell. Biochem. 65:259-266. © 1997 Wiley-Liss, Inc.
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  • 67
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    Journal of Cellular Biochemistry 68 (1998), S. 128-137 
    ISSN: 0730-2312
    Keywords: oligodendrocytes ; cell cycle ; differentiation ; cyclin-dependent kinases ; cdk5 ; cdk2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Oligodendrocytes, the myelinating cells of the central nervous system, are terminally differentiated cells that originate through asynchronous waves of proliferation and differentiation of precursors present at birth. Withdrawal from cell cycle and onset of differentiation are tightly linked and depend on an intrinsic program modulated by the action of growth factors. p27 plays a central and obligatory role in the initiation of oligodendrocyte differentiation and cessation of proliferation. In this paper, we have characterized the role of modulation of cdk2 and cdk5 kinase activity during the process of oligodendrocyte precursor differentiation. As rat primary oligodendrocytes differentiate in culture there is a fall in cdk2 activity and a rise in cdk5 activity as well as an increase in the cdk inhibitor, p27 protein. The decline in cdk2 activity is not accompanied by a drop in cdk2 protein level, suggesting that it results from inhibition of cdk2 activation rather than decreased protein expression. Taken together, these data suggest that oligodendrocytes may withdraw from the cell cycle at G1-S transition through inactivation of cdk2 activity, possibly initiated by increasing amount of p27, and that cdk5 may have a role until now unrecognized in the differentiation of oligodendrocytes. J. Cell. Biochem. 68:128-137, 1998. © 1998 Wiley-Liss, Inc.
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  • 68
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    Journal of Cellular Biochemistry 68 (1998), S. 411-426 
    ISSN: 0730-2312
    Keywords: bone marrow stroma ; human ; differentiation ; TGF-β ; BMP-2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β2) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β2 increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β2 regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β2 led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β2 attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β2-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β2 on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β2 response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β2 did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β2 acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β2 and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411-426, 1998. © 1998 Wiley-Liss, Inc.
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  • 69
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    Journal of Cellular Biochemistry 69 (1998), S. 169-180 
    ISSN: 0730-2312
    Keywords: growth factor ; bone ; osteoblast ; inflammation ; alkaline phosphatase ; differentiation ; proliferation ; PDGF ; mineralized nodules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Platelet-derived growth factor (PDGF) stimulates chemotaxis and proliferation of osteoblasts, and induces bone formation in vivo. To determine how PDGF might regulate these cells, the effect of PDGF on long-term mineralizing cultures of fetal rat osteoblastic cells was examined. Although PDGF increased cell proliferation in these cultures, continuous treatment with PDGF caused a dose-dependent decrease in mineralized nodule formation. When cells were treated with multiple, brief (1 day) exposures to PDGF at the osteoblast differentiation stage, there was a significant 50% increase in mineralized nodule area. Based on modulation of alkaline phosphatase activity it appears that longer-term exposure to PDGF reduces mineralized nodule formation largely by inhibiting differentiated osteoblast function, while short-term exposure enhances proliferation without inhibiting the differentiated phenotype. Thus, the ultimate affect of PDGF on bone formation is likely to reflect two processes: a positive effect through enhancing cell number or a negative effect by inhibiting differentiated function. The inhibitory effect of PDGF on formation of a mineralized matrix is unlikely to be simply a result of enhanced proliferation of “fibroblastic” cells since cultures treated with PDGF for 3 days and then transferred to new plastic dishes exhibited a 70% increase in mineralized nodule area compared to controls. These results would predict that multiple, brief exposures to PDGF would enhance bone formation in vivo, while prolonged exposure to PDGF, which is likely to occur in chronic inflammation, would inhibit differentiated osteoblast function and limit bone regeneration. J. Cell. Biochem. 69:169-180, 1998. © 1998 Wiley-Liss, Inc.
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  • 70
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    Journal of Cellular Biochemistry 70 (1998), S. 425-432 
    ISSN: 0730-2312
    Keywords: nerve growth factor ; tyrosine kinase receptors ; differentiation ; PC12 cells ; mitogen-activated protein kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Activation of receptor tyrosine kinases stimulates a diverse array of cellular responses such as proliferation and differentiation. The first events in the signal transduction pathways mediated by different receptor tyrosine kinases are similar and include activation of the mitogen-activated protein kinase (MAPK) pathway and the induction of immediate early genes. The precise signaling pathways leading to each of the cellular responses mediated by receptor tyrosine kinases are still unknown, although it has been proposed that sustained activation of the MAPK pathway by receptor tyrosine kinases such as the nerve growth factor (NGF) receptor TrkA is sufficient to induce differentiation in PC12 cells. In the present study we examined the effect of NGF on mutant PC12 cells that were derived spontaneously in our cultures. NGF induced normal activation of immediate early genes in these cells, whereas the activation of some delayed response genes, as well as neurite outgrowth, was impaired. Furthermore, activation of the NGF-induced extracellular signal-regulated kinase (ERK) in these cells was transient, not sustained. These results support the hypothesis that sustained activation of ERK plays an important role in activating the induction of delayed response genes. However, sustained ERK activation is not a mandatory condition for the promotion of all the features of differentiated PC12 cells, as NGF could induce transcription of the delayed response gene, transin, in PC12 mutant cells. Taken together, our results suggest that NGF induces differentiation of PC12 cells via several signaling pathways, an important one of which is the MAPK pathway. J. Cell. Biochem. 70:425-432, 1998. © 1998 Wiley-Liss, Inc.
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  • 71
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    Journal of Cellular Biochemistry 49 (1992), S. 37-45 
    ISSN: 0730-2312
    Keywords: differentiation ; osteocalcin ; osteoblast ; vitamin D ; responsive element ; promoter elements ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vitamin D is a physiological regulator of gene transcription associated with control of a broad spectrum of biological processes that include but is not restricted to growth, differentiation and calcium-mediated homeostatic control. Transcriptional regulation is mediated by sequence-specific interactions of a 1,25(OH)2D3-vitamin D receptor-accessory factor complex with vitamin D responsive elements (VDRE) residing in the promoters of hormone responsive genes. Functioning primarily as a transcription enhancer, activity at the VDRE is controlled by diverse and integrated cellular signalling pathways acting synergistically and/or antagonistically with a series of basal regulatory elements and other hormone regulated sequences that are components of modularly organized vitamin D-responsive gene promoters. Molecular mechanisms that integrate the activities at promoter elements contributing to vitamin D-related transcriptional control include overlapping transcription factor binding domains within regulatory elements and cooperative activities at independent regulatory sequences that determine the level of vitamin D responsiveness.
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  • 72
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    Journal of Cellular Biochemistry 49 (1992), S. 219-224 
    ISSN: 0730-2312
    Keywords: differentiation ; transgenic ; transcription factor ; C/EBP, obesity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5′-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and transacting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.
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  • 73
    ISSN: 0730-2312
    Keywords: bistratene A ; phorbol ester ; bryostatin ; protein kinase C ; differentiation ; phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bistratene A, a polyether toxin isolated from the colonial ascidian Lissoclinum bistratum, causes incomplete differentiation of human leukemia (HL-60) cells apparently through a mechanism not involving protein kinase C. In view of the importance of phosphorylation/dephosphorylation in cellular growth and differentiation we have investigated protein phosphorylation in these cells following exposure to bistratene A, using two-dimensional polyacrylamide gel electrophoresis. Marked increases in the phosphorylation of a protein of 20 kDa, pl 6.7, and a basic protein of 25 kDa were observed after incubation with bistratene A. A comparison was made with cells treated with 12-O-tetradecanoylphorbol 13-acetate and bryostatin 5. While changes in phosphorylation patterns were observed with these two compounds, the 20 kDa and 25 kDa proteins did not undergo phosphorylation changes. The 20 kDa protein was induced rapidly by very low concentrations of bistratene A reaching near maximal levels with 10 nM at 15 min exposure. This protein was found to be localised to the cytoplasm. Phosphoaminoacid analysis demonstrated that the majority of 32P was present in serine and tyrosine residues. The increased phosphorylation of the 20 kDa protein appeared to be due to hyperphosphorylation of existing protein although there was some increase in the amount of the protein. These results suggest that bistratene A will be a useful tool with which to investigate cellular differentiation mechanisms.
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  • 74
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    Journal of Cellular Biochemistry 21 (1983), S. 63-75 
    ISSN: 0730-2312
    Keywords: neuroblastoma ; differentiation ; EGF ; binding assay ; K+ transport ; Na+ transport ; amiloride ; growth stimulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown that exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+ -K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.
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  • 75
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    Journal of Cellular Biochemistry 22 (1983), S. 245-249 
    ISSN: 0730-2312
    Keywords: differentiation ; nuclear proteins ; maturation ; DMF ; polar solvents ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A human colonic carcinoma cell line was exposed to a nontoxic concentration of N,N-dimethylformamide (DMF) for 2 wk. Nuclear proteins were isolated from control and treated cells and compared by sodium dodecyl sulfate (SDS) electrophoresis. Qualitative and quantitative differences were observed. Metabolic labeling with tritiated leucine demonstrated qualitative variation between control and treated cells.
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  • 76
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    Journal of Cellular Biochemistry 26 (1984), S. 75-81 
    ISSN: 0730-2312
    Keywords: actyl-CoA carboxylase ; HL-60 ; differentiation ; leukemia ; fatty acid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In order to better understand the mechanism by which changes in the fatty acid composition of cellular lipids occur in leukemia cell lines induced to differentiate, the activity of the first enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase (EC 6.4.1.2) was measured in HL-60 promyclocytic leukemia cells before, during and after treatment with compounds that induce these cells to mature to neutro-phillike cells. After 24 h of exposure to dimethylsulfoxide, retinoic acid, or butyric acid, no morphological or biochemical (nitroblue tetrazolium reduction) evidence of differentiation occurred, but acetyl-CoA carboxylase activity decreased 44, 44.5, and 49% respectively, compared to untreated cells. After 7 days of culture in the presence of these agents, 79, 83, and 72% of cells acquired the ability to reduce nitroblue tetrazolium (versus 15% of control cells) and enzyme activity decreased 92.7, 99.7, and 98%, compared to control cultures, with the three compounds respectively. Thus, some of the reported changes in fatty acid composition of leukemia cells with differentiation may arise, in part, from the depression of the de novo fatty acid biosynthetic pathway and the loss of acetyl-CoA carboxylase activity may be a useful marker for neutrophilic differentiation in HL-60 cells.
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  • 77
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    Journal of Cellular Biochemistry 39 (1989), S. 443-454 
    ISSN: 0730-2312
    Keywords: proliferation ; differentiation ; growth factor receptors ; embryogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A receptor for fibroblast growth factor (aFGF, bFGF) was partially characterized in intact cell cultures, cell plasma membranes, and tissue plasma membrane preparations. Analysis of 24 different cell types from four species identified a 165-kDa FGF receptor present on the cell surface of most mesodermal and neuroectodermal cells. Chemical crosslinking of 125I-aFGF to its cell surface receptor was specifically blocked by a 100-fold molar excess of either aFGF or bFGF. In contrast to the similar molecular weight of FGF receptors, different cell types exhibited significant variations in binding of 125I-aFGF to intact cultures with low values of 8 pM and 700, to high values of 60 pM and 30,000, for the Kd and receptor number per cell, respectively. A binding assay was developed for quantitation of 125I-aFGF binding to cell- and tissue-derived membrane preparations. Membranes prepared from baby hamster kidney cells exhibited a Kd of 55 pM, while a similar Kd of 67 pM was determined for intact baby hamster kidney cells. Although ten different adult bovine tissue membrane preparations and human term placental membranes exhibited no specific binding of 125I-aFGF, FGF receptor was detected in embryonic murine tissues (17 days gestation). These results support the existence, in a variety of cells, of either a common FGF receptor that binds both aFGF and bFGF or closely related FGF receptors that cannot be distinguished by molecular weight. The differential binding of FGF to its receptor in embryonic vs. adult tissues suggests a potentially broad role for FGF in embryonic development and a more restrictive role in the adult.
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  • 78
    ISSN: 0730-2312
    Keywords: prolongation of G1 ; activin A ; RB protein ; cell cycle ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF-β1 family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K652 cells. Activin phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell, except that there is prolongation of the G1 phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1 is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9-24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle-related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1 phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells. © 1992 Wiley-Liss, Inc.
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  • 79
    ISSN: 0021-9304
    Keywords: polyelectrolyte complex ; human periodontal ligament fibroblast ; morphology ; proliferation ; differentiation ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: Human periodontal ligament fibroblast (HPLF) cultured on tissue culture dishes (TCD), irrespective of the presence of serum, showed only a spreading form. In contrast, using polyelectrolyte complex (PEC) as a matrix, HPLF showed spreading, round, and aggregate forms. Cells of the inner part of the aggregate contacted with each other to form a three-dimensional structure, and this condition corresponded to typical tissues in vivo. These seemed to be related to the interrelation between growth and morphology; that is, the HPLF of the spreading form was considered to belong to a proliferation phase, and the HPLF of the round and aggregate forms, with a little growth, seemed to belong to a functional phase of the cell cycle, indicating that PEC is able to control such cell functions as proliferation, morphology, and differentiation. The cell aggregate was observed only on PEC with carboxymethyl residues and was stained by alizarin red (AR), which suggested mineralization. The spreading cells on PEC containing sulfate residues were not stained by AR. Therefore, it was found that there was a certain relationship between cell growth and morphology, and that PEC affected the cell cycle and promoted proliferation and differentiation of HPLF. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 41, 257-269, 1998.
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  • 80
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    Journal of Biomedical Materials Research 37 (1997), S. 508-516 
    ISSN: 0021-9304
    Keywords: osteogenesis ; proliferation ; differentiation ; osteoblast ; human ; hydroxyapatite ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: In order to evaluate whether human osteoblastic cells differentiate normally on hydroxyapatite, we have compared the adhesion, proliferation, and differentiation of human trabecular (HT) osteoblastic cells on synthetic-dense hydroxyapatite and on standard plastic culture. We show here that initial HT cell attachment was 4-fold lower on hydroxyapatite than on plastic after 4 h of culture, and that normal cell attachment on hydroxyapatite was restored after 18 h of culture. HT cell proliferation was similar on the two substrates at 2-8 days of culture, but was lower on hydroxyapatite compared to plastic after 15 and 28 days of culture, as evaluated by DNA synthesis or cell number. HT cells cultured on both substrates produced an abundant extracellular matrix which immunostained for Type I collagen. The levels of carboxyterminal propeptide of Type I procollagen (P1CP) in the medium were lower in HT cell cultures on hydroxyapatite than on plastic. In addition, (3H)-proline incorporation into matrix proteins and the mean thickness of matrix layers were 52% and 26% lower, respectively, on hydroxyapatite compared to plastic after 4 weeks of culture, indicating that the total collagenous matrix synthesized by HT cells was lower on hydroxyapatite. However, (3H)-proline and calcium uptake expressed per cell was higher on hydroxyapatite than on plastic. The results show that human osteoblastic cells attach, proliferate, and differentiate on dense hydroxyapatite with a sequence similar to that of plastic. However, the growth of human osteoblastic cells is lower on hydroxyapatite in long-term culture, which results in a reduced amount of extracellular matrix, although matrix production per cell may be increased. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 508-516, 1997.
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  • 81
    ISSN: 0021-9304
    Keywords: implant ; titanium ; osteoblasts ; surface roughness ; 1α,25- (OH)2D3 ; differentiation ; local factor ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: Surface roughness has been shown to affect differentiation and local factor production of MG63 osteoblast-like cells. This study examined whether surface roughness alters cellular response to circulating hormones such as 1α,25-(OH)2D3. Unalloyed titanium (Ti) disks were pretreated with HF/HNO3 (PT) and then were machined and acid-etched (MA). Ti disks also were sandblasted (SB), sandblasted and acid etched (CA), or plasma sprayed with Ti particles (PS). The surfaces, from smoothest to roughest, were: PT, MA, CA, SB, and PS. MG63 cells were cultured to confluence on standard tissue culture polystyrene (plastic) or the Ti surfaces and then treated for 24 h with either 10-8M or 10-7M 1α,25-(OH)2D3 or vehicle (control). Cellular response was measured by assaying cell number, cell layer alkaline phosphatase specific-activity, and the production of osteocalcin, latent (L) TGFβ, and PGE2. Alkaline phosphatase activity was affected by surface roughness; as the surface became rougher, the cells showed a significant increase in alkaline phosphatase activity. Addition of 1α,25-(OH)2D3 to the cultures caused a dose-dependent stimulation of alkaline phosphatase activity that was synergistic with the effect caused by surface roughness alone. 1α,25-(OH)2D3 also caused a synergistic increase in osteocalcin production as well as local factor (LTGFβ and PGE2) production on the rougher CA, SB, and PS surfaces, but it had no effect on the production on smooth surfaces. The inhibitory effect of surface roughness on cell number was not affected by 1α,25-(OH)2D3 except on the SB surface. 1α,25-(OH)2D3 decreased cell number, increased alkaline phosphatase activity and osteocalcin production, and had no effect on LTGFβ or PGE2 production by MG63 cells grown on tissue culture polystyrene. These data suggest that bone cell response to systemic hormones is modified by surface roughness and that surface roughness increases the responsiveness of MG63 cells to 1α,25-(OH)2D3. They also suggest that the endocrine system is actively involved in normal bone healing around implants. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 39, 77-85, 1998.
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  • 82
    ISSN: 0021-9304
    Keywords: implant ; titanium ; osteoblasts ; prostaglandin ; indomethacin ; surface roughness ; 1α,25-(OH)2D3 ; differentiation ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: Surface roughness affects proliferation, differentiation (alkaline phosphatase and osteocalcin), local factor production [transforming growth factor (TGFβ) and prostaglandin E2 (PGE2)], and response to 1,25-(OH)2D3 (1,25) of MG63 osteoblast-like cells. In this study, we examined whether the effect of surface roughness on MG63 cells is mediated by prostaglandins produced by the cells. Unalloyed titanium (Ti) disks were pretreated with HF/HNO3 (PT) and then machined and acid-etched (MA). Disks were also coarse grit-sandblasted (SB), coarse grit-sandblasted and acid-etched (CA), or plasma-sprayed with Ti particles (PS). The surfaces, from smoothest to roughest, were PT, MA, CA, SB, and PS. MG63 cells were cultured to confluence on the Ti disks in the presence or absence of 10-7M indomethacin (Indo), a specific inhibitor of cyclooxygenase activity, resulting in decreased prostaglandin production. When the cells reached confluence, cell number, cell layer alkaline phosphatase specific activity (ALPase), and osteocalcin (OC) and latent TGFβ (LTGFβ) production were determined. In addition, confluent cultures which had been grown in the absence of Indo were exposed to 10-7M 1,25, 10-7M Indo, or a combination of the two for 24 h. On the rougher surfaces, cell number was decreased and ALPase, OC, and LTGFβ were increased. When indomethacin was present throughout the culture period, the effect of surface roughness on cell number, OC, and LTGFβ was abolished. ALPase was reduced, but surface roughness-dependent effects were still observed. Addition of indomethacin to confluent cultures for 24 h had no effect on any of the parameters examined, with one exception: Cells cultured on MA surfaces exhibited a more differentiated phenotype. 1,25 increased all parameters examined on SB, CA, and PS surfaces. When indomethacin was added with 1,25, the 1,25-dependent effects on cell number and OC and LTGFβ production were abolished; however, ALPase was unaffected. This indicates that bone cell response to systemic hormones may be modified by implant surface roughness. This effect may be mediated, at least in part, by prostaglandins produced by the same cells. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 41, 489-496, 1998.
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  • 83
    ISSN: 0021-9304
    Keywords: alginate beads ; in vitro ; chondrocytes ; differentiation ; biomaterials ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: Chondrocytes from 21-day-old rat fetal nasal cartilage were cultured in alginate beads for up to 20 days. It was found that chondrocytes retained their spherical shape and typical chondrocytic appearance. During the culture time, chondrocytes underwent differentiation, as demonstrated by the alkaline phosphatase-specific activity and rate of proteoglycan synthesis. Morphological data confirmed chondrocyte differentiation with the appearance of hypertrophic chondrocytes scattered in the alginate gel and a dense extracellular matrix containing filamentous structures and matrix vesicles. In addition, Northern blot analysis performed on day 8 of culture showed that chondrocytes cultured in alginate beads expressed type II collagen mRNA. The alginate bead method also appeared to be suitable for testing biomaterials, and the ready dissolution of the alginate beads by chelating agents provided a simple means for the rapid recovery of encapsulated chondrocytes. Powdered glass-ceramic particles entrapped in the alginate gel were colonized by chondrocytes, which then proliferated and formed a tissue similar to a true calcified cartilaginous structure. These results indicate that the alginate system represents a relevant model for studies of chondrogenesis and endochondral ossification. Furthermore, the encapsulation method could prove useful for studies of tissue-biomaterial interactions in an in vitro environment which more closely mirrors the cartilage matrix than other culture methods. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 42, 213-222, 1998.
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  • 84
    ISSN: 0021-9304
    Keywords: polyelectrolyte complex ; human periodontal ligament fibroblast ; proliferation ; differentiation ; alkaline phosphatase ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: In addition to many types of extra cellular matrix (ECM) in vivo, cells are stimulated by many types of vitamins, hormones, growth factors, etc. In this paper the effects of L-ascorbic acid 2-phosphate (Asc-2P) and dexamethasone (Dex) on proliferation and differentiation of human periodontal ligament fibroblast (HPLF) using polyelectrolyte complex (PEC) as a matrix in vitro will be discussed. The PEC was composed of chitosan as a polycation, with carboxymethyl (CPEC) or sulfated chitin (SPEC). Asc-2P (0.2 mM) inhibited the growth of HPLF on CPEC, but promoted the growth on SPEC. Moreover, the aggregation of HPLF on CPEC was inhibited by Asc-2P, but that on SPEC was induced in the presence of Asc-2P and Dex. Although Asc-2P reduced an increase in alkaline phosphatase (ALPase) activity of HPLF on CPEC as well, it induced a twofold increase in ALPase activities on SPEC and TCD. Furthermore, in the medium containing Asc-2P and 100 mM of Dex, cell growth was inhibited, but ALPase activity was promoted on both SPEC and TCD to form many aggregates on SPEC. ALPase activity increased by twofold over that of HPLF cultured in the medium containing only Asc-2P. Therefore, it is suggested that the cell functions of HPLF are controlled by the combination of PEC and additives. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 41, 270-277, 1998.
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  • 85
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    Journal of Cellular Physiology 102 (1980), S. 11-18 
    ISSN: 0021-9541
    Keywords: ornithine decarboxylase ; polyamines ; antizyme ; muscle ; myoblast ; differentiation ; proliferation ; density-dependent growth ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Four different approaches were used to test a postulated relationship between ornithine decarboxylase (ODC) activity and L6 muscle cell proliferation. In the first approach, the level of ODC activity in myoblasts was compared to that of myotubes. The fusion of proliferating myoblasts to form postmitotic myotubes is a unique property of skeletal muscle differentiation which offers special advantages for this study. If ODC activity reflects proliferation, it should be reduced in myotubes. Four hours after the addition of 10% horse serum (HS), ODC activity in myoblasts was 140 times that in myotubes. The small amount of ODC activity measured in myotube cultures could be attributed to remaining myoblasts. In the second approach, cell density was varied to alter the rate of cell division. Density-dependent inhibition of myoblast proliferation was closely correlated with a decrease of ODC activity. In the third approach, HS concentration was varied to alter the rate of cell division. Through a tenfold change of HS concentration, ODC activity paralleled cell proliferation. In the fourth approach, 1mM putrescine or spermidine was added with 10% HS to myoblasts. Although these treatments prevented the increase in ODC in response to HS, cell division was unimpaired; it seems likely that the polyamines entered the cell and substituted for endogenous polyamines. Mixing experiments indicated the presence of an inhibitor, tentatively identified as ODC antizyme, in homogenates of polyamine-treated cells. We conclude that ODC activity closely reflects proliferation in muscle cells, although the presence of extracellular polyamines may disrupt this association.
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  • 86
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    Journal of Supramolecular Structure 11 (1979), S. 503-515 
    ISSN: 0091-7419
    Keywords: glycoproteins ; two-dimensional electrophoresis ; differentiation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The present work examined the expression of cell surface glycoprotein antigens in cultured human cell lines. The set of glycoproteins studied was defined by their immunoreactivity with antiserum developed to Triton-solubilized extracts of placental brush border membranes. Studies were performed using cell lines of trophoblastic (BeWo, JEG-3) and nontrophoblastic (Chang liver cells) origin, as well as diploid fibroblast cell lines (WI-38, GM-38).Antiplacental brush border antiserum reacts with at least 19 distinct antigens present in placental membrane preparations, each of which can be resolved and identified in two-dimensional electrophoresis. The subunit molecular weight and isoelectric point for all components were defined by their positions in the two-dimensional matrix. Thirteen of these could be detected among the five cell lines examined by lactoperoxidase-catalyzed cell surface iodination. One of these 13 antigens has been identified as the placental isoenzyme of alkaline phosphatase (PAP). The expression of this component is limited to choriocarcinonia cells and Chang liver cells and it is not present in diploid fibroblasts. Under normal circumstances expression of PAP is unique to the differentiated placenta but has been frequently demonstrated in both trophoblastic and nontrophoblastic neoplasms.Two other antigens are variably expressed among the different cell types examined in the present study and their presence or absence was independent of the trophoblastic, epithelial nontrophoblastic, or fibroblastic origin of the cells.Ten surface antigens were expressed in all five cell lines. Six of these had previously been found common to membranes from three adult differentiated tissues, including liver and kidney, as well as placenta (Wada et al, J Supramol Struc 10(3):287-305, 1979). The presence of this set of antigens in cultured cells as well extends the possibility that these are ubiquitously expressed on human cell surfaces. Two other antigens observed in all cultured cells had been found in both placental and either kidney or liver membranes and may represent common functions shared by many tissues which are also necessary for growth in vitro. The two remaining placental antigens seen in all cultured cells have previously been shown to be absent in adult tissues. Their presence in cultured cells but not in the membranes of resting differentiated tissues may signify the expression of glycoproteins characteristic of trophoblasts in all cells adapted to growth in culture.
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  • 87
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    Journal of Supramolecular Structure 12 (1979), S. 259-272 
    ISSN: 0091-7419
    Keywords: epithelial transport ; differentiation ; cyclic AMP ; cryoprotective solvents ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Rat mammary (Rama 25) and dog kidney (MDCK) epithelial cell cultures formed ‘domes’ of cells due to fluid accumulation in focal regions between the culture dish and the cell monolayer. Addition of ouabain caused collapse of domes, suggesting that transport functions were required for maintenance of domes.Dome formation in both epithelial cell lines was stimulated by a broad spectrum of known inducers of erythroid differentiation in Friend erythroleukemia cells. Among these inducers were: (1) polar solvents such as dimethylsulfoxide, dimethylformamide, and hexamethylene bisacetamide; (2) purines such as hypoxanthine, inosine, and adenosine; (3) low-molecular-weight fatty acids such as n-butyrate; and (4) conditions expected to elevate levels of cyclic AMP. In the latter group were activators of adenylate cyclase such as cholera toxin and prostaglandin E1; cyclic AMP phosphodiesterase inhibitors such as theophylline and 1-methyl-3-isobutylxanthine; and analogs of cyclic AMP.Induction of domes occurred 15-30 h after addition of inducer to the culture medium. Induction by chemicals was serum-dependent and required protein synthesis but not DNA synthesis. Induced dome formation was reversible after removal of inducer, requiring the contiuous presence of inducer. Reversal was also observed after either removal of serum or addition of inhibitors of protein synthesis.These results suggest the hypothesis that domes arise in these epithelial cultures by a process that is similar to cell differentiation and is influenced by cyclic AMP.
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  • 88
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    Journal of Supramolecular Structure 8 (1978), S. 431-438 
    ISSN: 0091-7419
    Keywords: Friend cells ; Na+/K+ ATPase ; K+ ion levels ; transport changes ; differentiation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Treatment of Friend erythroleukemia cells with several different chemical agents causes an early decrease in the 86Rb+ influx mediated by Na+/K+ adenosine triphosphatase (ATPase). These agents, which induced Friend cells to differentiate, include dimethylsulfoxide (DMSO), ouabain, hypoxanthine, and actinomycin D. The magnitude of the early decrease in 86Rb+ influx correlates with the proportion of cells in cultures of inducible Friend cell clones which later go on to synthesize hemoglobin. Compounds which do not incude differentiation in these cells, such as xanthine, exogenous hematin, and erythropoietin, do not cause a change in 86Rb+ influx. A change in the intracellular K+ ion concentration does not occur during induction by DMSO because, although there is a decrease in K+ content per cell soon after induction, there is a parallel decrease in cell volume. These results and previous observations from this laboratory are discussed in terms of the posible involvement of the Na+/K+ ATPase in Friend cell differentiation.
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  • 89
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    Journal of Supramolecular Structure 13 (1980), S. 513-524 
    ISSN: 0091-7419
    Keywords: long-term marrow cultures ; cell-cell interactions ; microenvironment ; stem cell ; proliferation modulators ; GM-CFC ; differentiation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In long-term marrow cultures haemopoiesis can be maintained in vitro for up to 6 months. Critical analysis of the cell populations produced has shown that the stem cells and their committed progeny have characteristics in common with the corresponding cell types in vivo. The maintenance of haemopoiesis in vitro is associated with the development of an appropriate inductive environment provided by bone marrow derived adherent cells. Analysis of the interactions between environmental and haemopoietic cells has been facilitated by the development of in vitro systems reproducing the naturally occurring genetic environmental defects and other systems where the development of a competent inductive environment shows a dependency upon corticosteroid hormones. Investigations have shown that stem cell proliferation may be controlled by production of opposing activities, one stimulatory for DNA synthesis, the other inhibitory. A model is proposed whereby modulation in the production of these factors is determined by the physical presence of stem cells in a proposed cellular milieu, within the adherent layer. The adherent layer, apart from acting at the level of stem cell proliferation, can also modify the response of differentiating cells (eg, GM-CFC) to exogenous stimulatory activities. Addition of GM-CSF or of CSF-antiserum has no effect on haemopoiesis in long-term cultures.
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  • 90
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    Journal of Supramolecular Structure 13 (1980), S. 339-372 
    ISSN: 0091-7419
    Keywords: extracellular matrix ; FGF ; vascular endothelial cells ; vascular smooth muscle cells ; aging ; differentiation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest.
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  • 91
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    Cell Biochemistry and Function 1 (1983), S. 92-96 
    ISSN: 0263-6484
    Keywords: Blood cells ; leukaemia ; myelodysplasia ; cytochemistry ; neutrophils ; microdensitometer ; lysosomes ; preleukaemia ; maturation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Quantitative cytochemistry of components of blood neutrophil azurophilic granules (myeloperoxidase, chloroacetate esterase, β-glucuronidase, and acid phosphatase) and specific granules (lactoferrin) has been performed by scanning and integrating micro-densitometry in 13 patients with a myelodysplastic syndrome and 11 patients with chronic granulocytic leukaemia. Both patient groups showed a reduction of enzyme activity in azurophilic granules, and also of lactoferrin, consistent with abnormal development of neutrophil granules. These cytochemical changes in blood neutrophils are similar to those found in acute myeloid leukaemia, are consistent with a leukaemic maturation defect, and may be of diagnostic value.
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  • 92
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    Cell Biochemistry and Function 1 (1983), S. 131-140 
    ISSN: 0263-6484
    Keywords: Biochemistry ; polyamines ; putrescine ; spermidine ; spermine ; ornithine decarboxylase ; biosynthesis ; cell proliferation ; oxidized polyamines ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The naturally-occurring polyamines exist in the free form, as N-acetyl derivatives and bound to protein. Their biosynthesis is subject to sensitive control, particularly of ornithine decarboxylase. This enzyme may be multifunctional and a key regulatory protein. Studies, principally with selective inhibitors, have elucidated the roles of polyamines in cell proliferation. Oxidized polyamines, in contrast, can be potent mitotic inhibitors. These effects are reviewed in terms of their chemistry and biochemistry. Their principal distinctions are that they can be made or degraded intracellularly, they can associate electrostatically with macromolecules by means of their spaced cationic groups, and these can be readily converted to covalent bonds.
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  • 93
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    Cell Biochemistry and Function 6 (1988), S. 7-12 
    ISSN: 0263-6484
    Keywords: Aging ; ethanol metabolism ; alcohol dehydrogenase ; microsomal functions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The study of the influence of the age of the animals (13 to 53 weeks) on the rate of ethanol metabolism in vivo and the total activity of liver alcohol dehydrogenase and microsomal ethanol oxidizing system showed a progressive decline with age. These effects were observed concomitantly with a diminution in the content of cytochrome P-450 and microsomal functions related to oxidative and free-radical mediated reactions, namely, NADPH oxidase activity, NADPH-dependent oxygen uptake and NADPH-or t-butyl hydroperoxide-induced chemiluminescence. It is concluded that ageing is accompanied by a diminution in the total oxidative activity of the liver tissue, which would explain the depression in basal and ethanol-induced lipid peroxidation found in the oldest group of rats studied.
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  • 94
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    Cell Biochemistry and Function 1 (1983), S. 168-172 
    ISSN: 0263-6484
    Keywords: Bone ; aerobic glycolysis ; fatty acid oxidation ; cartilage ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The apparent paradox of aerobic glycolysis has been investigated in bone and in cartilage. A new cytochemical procedure for hydroxyacyl dehydrogenase (HOAD) activity showed that the maximal activity of this enzyme in both tissues was equivalent to the maximal activity of glyceraldehyde 3-phosphate dehydrogenase (GAPD). The sum of these activities gave a measure of the maximum amount of acetyl-coenzyme A that could be produced. In these tissues, but not in liver which does not exhibit aerobic glycolysis, this summed value exceeded the maximal activity of succinate dehydrogenase (SDH). Consequently, it suggested that where fatty acid oxidation is sufficient to supply all the acetyl-coenzyme A required for the Krebs' cycle, that derived from fatty acid oxidation may inhibit pyruvate dehydrogenase causing accumulation of pyruvate which must be converted to lactate if pentose-shunt activity is to be maintained.
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  • 95
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    Cell Biochemistry and Function 1 (1983), S. 173-178 
    ISSN: 0263-6484
    Keywords: RNA ; quantitative cytophotometry ; luteal regression ; ovarian 20α-hydroxypregn-4-ene-3-one ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Luteal and interstial cell RNA contents and circulating progesterone (P) and 20α-hydroxypregn-4-ene-3-one (20α-OH P) levels were measured during pseudopregnancy in order to characterize relationships between ovarian 20α-OH P secretion and luteal regression. Functional luteolysis, as manifested in depressed P levels, was not associated with concurrent elevations in 20α-OH P. Rather, augmented 20α-OH P levels were evidenced in periovulatory periods at the onset and termination of pseudopregnancy, subsequent to RNA accumulation in both luteal and interstitial compartments. It is postulated that 20α-OH P, the putative inactive metabolite of P, is produced by both ovarian compartments in a cyclic manner and in response to gonadotrophin released in the preovulatory period.
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  • 96
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    Cell Biochemistry and Function 1 (1983), S. 186-186 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 97
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    Cell Biochemistry and Function 1 (1983), S. 187-187 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 98
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    Cell Biochemistry and Function 1 (1983), S. 179-185 
    ISSN: 0263-6484
    Keywords: Blood ; forskolin ; protein phosphorylation ; platelets ; release reaction ; secretion ; cAMP ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of diterpene forskolin on the human platelet release reaction and on platelet protein phosphorylation were studied. This drug is shown to have the same effects as other agents which increase cAMP levels, namely, it inhibits the secretory response to diverse agonists and causes changes in the phosphorylation of several specific proteins. An increase of the 32P content is seen in the MW 47 000, 24 000 and 21 000 polypeptides while a decrease is observed in the MW 41 000 and 27 000 and 20 000 species. Forskolin also inhibits the changes in protein phosphorylation pattern induced by the powerful platelet secretagogue, thrombin. Our results relate the effects of antagonists of platelet secretion such as prostaglandins more closely to changes in cAMP levels and in protein phosphorylation than to other possible effects of the receptor-ligand interaction, which is by-passed by the use of forskolin. Our results also provide additional evidence involving these changes in the mechanisms which regulate the secretory process in platelets.
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  • 99
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    Cell Biochemistry and Function 8 (1990), S. 31-38 
    ISSN: 0263-6484
    Keywords: Proximal tubule ; Pi depletion ; calcium ; phorbol ester ; gluconeogenesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Pi depletion of proximal tubule cells isolated from mouse kidney results in a decrease in the cell content of fructose-2,6-bisphosphate and an increase in the rate of gluconeogenesis from pyruvate, malate and succinate. Gluconeogenesis from glycerol is unaffected by Pi depletion. Introduction of fructose-2,6-bisphosphate into the cytosol of ATP-permeabilized cells is accompanied by a fall in gluconeogenesis.The presence of external Ca2+ stimulates gluconeogenesis. When cytosolic Ca2+ is raised to 1·8 μM by permeabilization, the resealed cells still require 2·5 mM Ca2+ in the bathing medium in order to perform gluconeogenesis at the maximum rate. Cells permeabilized in the presence of cAMP show a decreased rate of glucose production. Phorbol ester stimulates gluconeogenesis provided that the phorbol treatment is performed in the absence of Ca2+ ions.It is suggested that Pi depletion may stimulate pyruvate carboxylase activity and facilitate the entry of certain gluconeogenic substrates into mitochondria. It is also proposed that important aspects of the control of renal gluconeogenesis by parathyroid hormone are mediated by protein kinase C.
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  • 100
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    Cell Biochemistry and Function 8 (1990), S. 39-47 
    ISSN: 0263-6484
    Keywords: Fructose-1, 6-diphosphate (FDP) ; heart slices ; GABA-receptors ; Ca++ entry ; Cl- up-take ; gastric ulcers ; serum transaminases ; diuresis ; narcosis ; dependence and withdrawal symptoms ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Fructose-1, 6-diphosphate (FDP) decreases the effect of ethanol on Ca++ entry and inhibits the ethanol-stimulated phosphate efflux in rat heart slices. FDP also inhibits the ethanol-stimulated [36Cl-]-uptake by rat brain microvesicles and affects the isolated GABA-receptor in a way opposite to that of ethanol. The in vivo effects of FDP include a dose-dependent decrease in ethanol-induced gastric ulcers and a decrease in the serum transaminase levels raised by chronic ethanol administration. Other central actions of ethanol such as diuresis, narcosis, dependence and withdrawal symptoms are also counteracted by FDP.
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