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  • Cell & Developmental Biology  (41,698)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 95-103 
    ISSN: 0730-2312
    Keywords: mechanical strain ; interleukin (IL)-α and β gene expression ; proliferation ; protein synthesis ; morphology ; keratinocyte biology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recent studies in our laboratory have demonstrated that mechanical strain alters many facets of keratinocyte biology including proliferation, protein synthesis, and morphology. IL-1 is known to play an important role in the autocrine regulation of these basic cellular properties under basal and stimulated conditions. However, it is not known whether IL-1 plays a role in strain-induced alteration of keratinocyte biology. Thus, the objective of this study was to test the hypothesis that cyclic strain stimulates IL-1 expression and that strain-induced changes in keratinocyte function is regulated by IL-1. To test this hypothesis, we examined the effect of cyclic strain (10% average deformation) on keratinocyte IL-1 gene expression and the effect of neutralizing antibodies of IL-1α and IL-1β on strain-induced changes in keratinocyte proliferation, morphology, and orientation. Northern blot analyses demonstrated that steady state levels of IL-1α and β mRNA were elevated by 4 h, peaked at 12 h of cyclic strain (IL-1α, 304 ± 14.2%; IL-1β, 212 ± 5.6% increase vs. static controls) and decreased gradually by 24 h. IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.01 μg/ml) significantly blocked strain-induced keratinocyte proliferation as well as the basal rate of proliferation. In contrast, IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.1 μg/ml) had no effect on strain-induced morphological changes such as elongation and alignment. We conclude that mechanical strain induces IL-1 mRNA expression in keratinocytes. The role of IL-1 in mediating strain-induced changes in keratinocyte biology remains to be determined but appears to be independent of morphological changes. J. Cell. Biochem. 69:95-103, 1998. © 1998 Wiley-Liss, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 104-116 
    ISSN: 0730-2312
    Keywords: mRNA export ; cell cycle ; gene transfection ; cultured mammalian cells ; hnRNP L ; nuclear transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human β-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless β-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes. J. Cell. Biochem. 69:104-116, 1998. © 1998 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 117-126 
    ISSN: 0730-2312
    Keywords: haemochromatosis gene ; histone gene cluster ; YACs ; cosmid contig ; sequences ; species comparison ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The HFE (HLA-H) gene is a strong candidate gene for hereditary haemochromatosis and was localized on the short arm of chromosome 6 to 6p21.3-p22. In addition, the sequence of the homologous mouse and rat cDNA and a partial sequence from the mouse gene have been reported recently. In this report, we describe the location of the human and the mouse HFE (HLA-H) gene within the histone gene clusters on the human chromosome 6 and the mouse chromosome 13. Both the human and the murine gene were located on syntenic regions within the histone gene clusters in the vicinity of the histone H1t gene. The genomic sequence of the human HFE (HLA-H) gene and the 3′ portion of the homologous mouse gene were determined. Comparison of the genomic sequences from man and mouse and the cDNA sequence from rat shows significant similarities, also beyond the transcribed region of the mouse gene. J. Cell. Biochem. 69:117-126, 1998. © 1998 Wiley-Liss, Inc.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 143-153 
    ISSN: 0730-2312
    Keywords: HB-EGF ; cleavage-secretion ; PKC ; ErbB1 ; EGF receptor ; matrix metalloproteinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143-153, 1998. © 1998 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 127-142 
    ISSN: 0730-2312
    Keywords: chromosome architecture ; disassembly ; reassembly ; proteases ; in vitro model ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Topoisomerase II has been suggested to play a major role in chromosome organization based on its DNA decatenating activity and its ability to mediate direct binding interactions between DNA and nuclear matrix. However, this latter point remains controversial. Here we address the question of whether the chromatin binding activity of Topoisomerase II is sufficient to modify chromosome form using whole mammalian chromosomes in vitro. Intact chromosomes were microsurgically removed from living cells and disassembled by treatment with protease or heparin. When these disassembled chromosomes were incubated with recombinant human Topoisomerase II, the enzyme became incorporated into chromatin and reassembly resulted, leading to almost complete restoration of pre-existing chromosome shape and position within minutes. Chromosome reconstituition by Topoisomerase II was dose-dependent, saturable, and appeared to be controlled stoichiometrically, rather than enzymatically. Similar reassembly was observed in the absence of ATP and when a catalytically inactive thermosensitive Topoisomerase II mutant was used at the restrictive temperature. Chromosome recondensation also could be induced after the strand-passing activity of Topoisomerase II was blocked by treatment with an inhibitor of its catalytic activity, amsacrine. When a non-hydrolyzable β,γ-imido analog of ATP (AMP-PNP) was used to physiologically fix bound Topoisomerase II enzyme in a closed form around DNA, subsequent chromosome disassembly was prevented in the presence of high salt. These data suggest that Topoisomerase II may control higher order chromatin architecture through direct binding interactions, independently of its well-known catalytic activity. J. Cell. Biochem. 69:127-142, 1998. © 1998 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 174-185 
    ISSN: 0730-2312
    Keywords: metallothionein ; isoform ; differential expression ; autoregulation ; Chinese hamster ovary cell ; cadmium-resistant cell ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transcription regulation of metallothionein (MT) isoform promoters was investigated in Chinese hamster ovary (CHO) K1 and MT gene amplified, cadmium-resistant (CdR) cells. The transfected promoter of Chinese hamster MTI and MTII genes can be activated in both cell lines by stimulation with Cd or Zn ions, although no MT mRNA can be detected in CHO K1 cells after challenge with metal ions. Neither MT promoter used in this study can be activated by induction with dexamethasone, regardless of whether a sequence homologous to glucocorticoid responsive element is present. During induction by metal ions, differential promoter activities of the MT genes occurs in both CHO K1 and CdR cells where MTII promoter has a stronger activity than that of MTI. As indicated by a time course study in both cell lines, the relative induction ratios of both MTI and MTII promoters are similar at each time interval. This result is consistent with a differential transcriptional factor-promoter interaction for the two MT promoters. By using the CHO K1 and CdR cells as a model system, the occurrence of autoregulation for yeast CUP1 (MT) gene was examined in mammalian cells. Both MT promoters consistently show a lower basal activity but a higher induction ratio in CHO K1 than CdR cells; a result different from that of yeast CUP1 gene. When MTF-1 mRNA was examined, no difference in relative quantity was observed in CHO K1 and in CdR cells treated with metal ions or with metal ions absent. J. Cell. Biochem. 68:174-185, 1998. © 1998 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    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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  • 8
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 154-168 
    ISSN: 0730-2312
    Keywords: transcription ; mRNA stability ; dexamethasone ; gene regulation ; glucocorticoid receptor ; rat calvarial osteoblasts ; osteopontin ; vitamin D receptor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts. J. Cell. Biochem. 69:154-168, 1998. © 1998 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 189-200 
    ISSN: 0730-2312
    Keywords: peroxisome proliferator activated receptor ; retinoid x receptor ; retinoic acid receptor ; liver hyperplasia ; hepatocarcinoma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Chronic griseofulvin (GF) feeding induces preneoplastic foci followed by hepatocellular carcinoma in the mouse liver. Our previous study suggested that GF-induced hepatocellular proliferation had a different mechanism from that of peroxisome proliferator (PP)-induced direct hyperplasia. The GF-induced hepatocellular proliferation was mediated through activation of immediate early genes such as Fos, Jun, Myc, and NFκB. In contrast, PP-induced direct hyperplasia does not involve activation of any of these immediate early genes. It has been shown that nuclear hormone receptors including peroxisome proliferator activated receptors (PPARs) and retinoid x receptors (RXRs) play important roles in mediating the pleiotropic effects of PPs. To examine the possible roles of PPARs and RXRs during non-PP-induced hepatocellular proliferation and the interaction between PP and non-PP-induced proliferation, we have studied the expression of the PPAR and RXR genes in the GF model using northern blot hybridizations and gel retardation assays. The data showed that the expression of PPARα and RXRα genes was down-regulated in the livers containing preneoplastic nodules and in the liver tumors induced by GF. The mRNA down-regulation was accompanied by a decrease in the amount of nuclear protein-bound to peroxisome proliferator and retinoic acid responsive elements. Down-regulation was also associated with the suppressed expression of the PPARα/RXRα target genes (i.e., acyl-Co oxidase and cytochrome P450 4A1) and the catalase gene. The RXRγ gene was also down-regulated, but the RARα, β, and γ and PPARβ and γ genes were up-regulated. These results indicated that the hepatocarcinogenesis induced by GF is accompanied by suppression of the PPARα/RXRα-mediated direct hyperplasia pathway. The differential expression of these nuclear hormone receptors reveals a new aspect for understanding the individual roles and intercommunication of PPAR, RXR, and RAR isoforms in the liver. J. Cell. Biochem. 69:189-200, 1998. © 1998 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 201-210 
    ISSN: 0730-2312
    Keywords: sodium butyrate ; alkaline phosphatase ; A5 cells ; A5-DAP cells ; A5-BAG cells ; β-galactosidase ; retroviral vectors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have studied the effects of sodium butyrate (NaBu) on the expression of genes transduced by retroviral vectors and stably expressed in two salivary gland-derived cell lines, A5-DAP and A5-BAG, established earlier. These cell lines were obtained by infecting A5 cells with the retroviral vectors DAP and BAG, respectively, and by selecting neomycin-resistant transduced cells. A5-DAP cells express human placental alkaline phosphatase (PLAP) and A5-BAG cells bacterial β-galactosidase, both under the control of the viral long terminal repeat (LTR) enhancer-promoter. NaBu in the concentration of 2-8 mM inhibited the growth of A5-DAP cells, and induced the expression of heat-stable PLAP. These effects of NaBu were dose-dependent. Induction of PLAP in clones of A5-DAP cells that express different basal levels of the enzyme was not correlated with the relative inducibilty by NaBu. Exposure to 4 mM NaBu for 48 h increased the PLAP mRNA level by 31%. A5-DAP cells released, in a time-dependent manner, PLAP into the culture medium. Cells treated with NaBu released more PLAP than untreated cells in proportion to their elevated level of the enzyme. The parent A5 cells also express a low level of tissue non-specific type alkaline phosphatase, which was also induced by NaBu. NaBu inhibited the growth of A5-BAG cells also, and increased the β-galactosidase level. These data indicate the genes transduced by retroviral vectors can be induced by NaBu, which most likely interacts with the viral LTR. J. Cell. Biochem. 69:201-210, 1998. © 1998 Wiley-Liss, Inc.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 211-220 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; protein kinase CK2 ; disulfide bonds ; sodium tetrathionate ; iodoacetamide ; sulfhydryl crosslinking ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nuclear matrix (NM) appears to be an intranuclear locale for significant and dynamic association of the ubiquitous multifunctional messenger-independent serine/threonine protein kinase CK2 that has been implicated in growth control [Tawfic et al. (1996): J Cell Biochem 61:165-171]. We have examined the nature of the association of CK2 with the NM. Nuclei prepared in the presence of a sulfhydryl-blocking reagent such as iodoacetamide demonstrate a reduction in the amount of CK2 associated with the NM to less than 5% of the control. On the other hand, when nuclei are treated with the sulfhydryl crosslinking reagent sodium tetrathionate, NM-associated CK2 increases severalfold. Treatment of nuclei with sodium tetrathionate followed by 2-mercaptoethanol blocks this increase. Nuclei isolated from rat liver and prostate behaved similarly, suggesting an identical mode of association of CK2 with the NM regardless of the organ. These results indicate a role of sulfhydryl interactions such that NM anchoring of CK2 occurs via its β subunit, which contains several vicinal cysteine residues. Further, various sulfhydryl-blocking reagents inhibited CK2 activity in a concentration-dependent manner, and the inhibitory effect was reversed by agents such as dithiothreitol, implying that cysteine residues in the CK2 play a role in its catalytic activity. J. Cell. Biochem. 69:211-220, 1998. Published 1998 Wiley-Liss, Inc.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 221-231 
    ISSN: 0730-2312
    Keywords: sodium fluoride ; stress response ; stress proteins ; heat shock proteins ; rat brain tumor 9L cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221-231, 1998. © 1998 Wiley-Liss, Inc.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 244-251 
    ISSN: 0730-2312
    Keywords: monomeric laminin receptor ; receptor maturation ; acylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule. J. Cell. Biochem. 69:244-251, 1998. © 1998 Wiley-Liss, Inc.
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  • 14
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 252-259 
    ISSN: 0730-2312
    Keywords: bisphosphonate ; prostaglandin F2α ; interleukin-6 ; phospholipase D ; osteoblast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In previous studies, we have reported that PGF2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein in osteoblast-like MC3T3-E1 cells, and that PGF2α and PGE1 induce interleukin-6 (IL-6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF2α- and PGE1-induced IL-6 synthesis in these cells. Tiludronate significantly suppressed the PGF2α-induced IL-6 secretion in a dose-dependent manner in the range between 0.1 and 30 μM. However, the IL-6 secretion induced by PGE1 or (Bu)2cAMP was hardly affected by tiludronate. The choline formation induced by PGF2α was reduced by tiludronate dose-dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF2α-induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP-binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF-induced IL-6 secretion in human osteoblastic osteosarcoma Saos-2 cells. These results strongly suggest that tiludronate inhibits PGF2α-induced IL-6 synthesis via suppression of phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP-binding protein and phospholipase D. J. Cell. Biochem. 69:252-259, 1998. © 1998 Wiley-Liss, Inc.
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  • 15
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 260-270 
    ISSN: 0730-2312
    Keywords: oncogenic function of mutant p53 ; MAR-DNA elements ; MAR-DNA binding by mutant p53 ; MethA p53 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We recently reported that murine MethA mutant but not wild-type p53 specifically binds to MAR-DNA elements (MARs) with high affinity. Here we show that this DNA binding activity is exerted not only by MethA mutant p53 but also by other murine mutant p53 proteins isolated from the transformed murine BALB/c cell lines 3T3tx and T3T3 and differing in their conformational status. High affinity MAR-DNA binding was not restricted to the XbaI-IgE-MAR-DNA fragment from the murine immunoglobulin heavy chain gene enhancer locus [Cockerill et al. (1987): J Biol Chem 262:5394-5397] used in previous studies, as MethA p53 also specifically interacted with other A/T-rich bona fide MARs. Not only murine but also human mutant p53 proteins carrying the mutational hot spot amino acid exchanges 175Arg→His, 273Arg→Pro, or 273Arg→His bound to the XbaI-IgE-MAR-DNA fragment. We therefore conclude that high affinity MAR-DNA binding is a property common to a variety of mutant p53 proteins. J. Cell. Biochem. 69:260-270, 1998. © 1998 Wiley-Liss, Inc.
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  • 16
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 282-290 
    ISSN: 0730-2312
    Keywords: estrogen modulation ; osteoblastic cells ; plasma membrane receptors ; nuclear receptors ; gap junction communication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Two osteoblastic cell populations, calvarial and marrow stromal cells, were exposed to estrogen derivatives in vitro. The hormonal effect was monitored by following intracellular Ca+2 levels [Ca+2]i and gap-junction communication. We measured fast changes in intracellular Ca+2 levels in response, of these cells, to the steroid hormones. The changes were dose dependent revealing maximal activity at 100 pM by 17-β-Estradiol and 1 nM by estradiol-CMO. Additionally, the effect of estrogen, on functional coupling of the cells, was measured using fluorescence dye migration and counting the number of neighboring cells coupled by gap junctions. An uncoupling effect was demonstrated in response of these cells to estrogen treatment. The quick stereospecific effect was achieved in the presence of 17-β-estradiol but not in the presence of 17-α-estradiol. These results suggest the involvement of plasma membrane receptors in addition to the already known nuclear receptors in transducing the hormone effects in the osteoblastic cells. J. Cell. Biochem. 69:282-290, 1998. © 1998 Wiley-Liss, Inc.
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  • 17
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 271-281 
    ISSN: 0730-2312
    Keywords: butyrate ; isobutyramide ; prostate cancer ; LNCaP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Progression to androgen independence remains the main obstacle to improving survival and quality of life in patients with advanced prostate cancer. Induction of differentiation may serve as a rational basis for prevention of progression to androgen independence by modulating gene expression activated by castration or upregulated during androgen-independent progression. The objectives of this study were to characterize the in vitro effects of sodium butyrate on human prostate cancer cell growth, PSA gene expression, and differentiation in the LNCaP tumor model and to determine whether tumor progression in vivo is delayed by isobutyramide, an orally bioavailable butyrate analogue with a longer half-life. The effects of isobutyramide on LNCaP tumor growth and serum PSA levels in both intact and castrate male mice were compared to controls. At concentrations 〉 1 mM, butyrate induced dose-dependent changes towards a more differentiated phenotype, G1 cell cycle arrest, and an 80% decrease in LNCaP cell growth rates. PSA gene expression was increased threefold by butyrate, indicative of differentiation-enhanced gene expression. The half-life of isobutyramide in athymic mice was determined by gas chromatography to be 4 h. During a 4 week period in intact-placebo mice, tumor volume and serum PSA increased 4.1- and 6.6-fold, respectively, compared to twofold and 2.7-fold increases in tumor volume and serum PSA in intact-treated mice. During a 7 week period in castrate-placebo mice, tumor volume and serum PSA levels increased 2.4-fold and fourfold, respectively, compared to a 50% reduction in tumor volume and a twofold increase in serum PSA above nadir levels in castrate mice treated with adjuvant isobutyramide. Isobutyramide treatment induced pronouced morphological changes in LNCaP tumor cells, with loss of defined nucleoli and dispersion of chromatin distribution. LNCaP tumor PSA mRNA levels actually increased threefold, indicative of differentiation-enhanced gene expression. This study demonstrates that butyrate causes LNCaP cell cycle arrest and increased PSA gene expression, both indicative of differentiation. The combination of castration and adjuvant isobutyramide was synergistic in delaying tumor progression. Decreased tumor cell proliferation and increased PSA gene expression induced by isobutyramide results in disconcordant changes in serum PSA and tumor volume and reduces the utility of serum PSA as a marker of response to therapy. J. Cell. Biochem. 69:271-281, 1998. © 1998 Wiley-Liss, Inc.
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  • 18
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 291-303 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; TGF-β1 ; bone ; osteoblast differentiation ; mineralization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4-6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β-treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β-treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β-dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291-303, 1998. © 1998 Wiley-Liss, Inc.
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  • 19
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    Journal of Cellular Biochemistry 69 (1998), S. 304-315 
    ISSN: 0730-2312
    Keywords: VAT-1 ; Pacific electric ray Torpedo californica ; ATPase ; Mus musculus ; gene structure ; Ehrlich ascites tumor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recently, interest has focused on the human gene encoding the putative protein homologous to VAT-1, the major protein of the synaptic vesicles of the electric organ of the Pacific electric ray Torpedo californica, after it has been localized on chromosome locus 17q21 in a region encompassing the breast cancer gene BRCA1. Chromosomal instability in this region is implicated in inherited predisposition for breast and ovarian cancer. Here we describe isolation and biochemical characterization of a mammalian 48 kDa protein homologous to the VAT-1 protein of Torpedo californica. This VAT-1 homolog was isolated from a murine breast cancer cell line (Ehrlich ascites tumor) and identified by sequencing of cleavage peptides. The isolated VAT-1 homolog protein displays an ATPase activity and exists in two isoforms with isoelectric points of 5.7 and 5.8. cDNA was prepared from Ehrlich ascites tumor cells, and the murine VAT-1 homolog sequence was amplified by polymerase chain reaction and partially sequenced. The known part of the murine and the human translated sequences share 97% identity. By Northern blots, the size of the VAT-1 homolog mRNA in both murine and human (T47D) breast cancer cells was determined to be 2.8 kb. Based on the presented data, a modified gene structure of the human VAT-1 homolog with an extended exon 1 is proposed. VAT-1 and the mammalian VAT-1 homolog form a subgroup within the protein superfamily of medium-chain dehydrogenases/reductases. J. Cell. Biochem. 69:304-315, 1998. © 1998 Wiley-Liss, Inc.
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  • 20
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    Journal of Cellular Biochemistry 69 (1998), S. 326-335 
    ISSN: 0730-2312
    Keywords: copper ; human endothelial cells ; angiogenesis ; growth factors ; cell proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 μM CuSO4 in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-α, transforming growth factor-β, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1α. Moreover, despite the previous observations that copper increased total specific binding of 125I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis. J. Cell. Biochem. 69:326-335, 1998. © 1998 Wiley-Liss, Inc.
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  • 21
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    Journal of Cellular Biochemistry 69 (1998), S. 316-325 
    ISSN: 0730-2312
    Keywords: osteoprogenitors ; mineralization ; marrow stroma ; Src ; tyrosine kinase dexamethasone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Src protein is essential for the regulation of bone turnover primarily via bone resorption because it is required in osteoclast differentiation and function. We followed temporal changes of Src protein abundance in marrow stromal cells induced to mineralize by dexamethasone (DEX), growth in cold temperature, or both. Given the tyrosine kinase function of Src and its numerous substrates, profiles of phosphotyrosine-containing proteins were followed as well. On day 11 of stimulation, specific alkaline phosphatase (ALP) activity at 30°C decreased under DEX relative to 37°C cultures, in accord with increased cell counts. Mineralization per well under DEX increased by 25% at 37°C, whereas at 30°C it increased by more than threefold regardless of the DEX stimulation. At 30°C, on a per cell basis mineralization increased 2.5 and 3 times with and without DEX, respectively. Cultures at 37°C showed a general drop per cell of many phosphotyrosine-containing proteins on day 3 relative to days 1 and 2 in both DEX-stimulated and nonstimulated cultures; several proteins did recover (recuperate) thereafter. On days 1 and 2, the phosphotyrosine signal was higher in several proteins under DEX stimulation; this trend became inverted after day 3. The changes in abundance per cell of Src protein (pp60src) followed a similar trend, and in addition a truncated Src molecule, p54/52src, was detected as a putative cleavage product presumably representing its carboxy terminus. The pp60src was most abundant, relative to its truncated product, in day 7 nonstimulated cultures, whereas under DEX stimulation the truncated species pp54/52src showed the highest relative abundance on days 7. At 30°C, DEX stimulation accentuated the increase in Src protein on day 3, showed no change on day 7, and returned to increase Src protein on day 10. Potassium ionophorvalinomycin, considered to select against mineralizing osteoprogenitors at 30°C, showed on day 10 in the absence of DEX a relative increase in truncated Src protein compared to both DEX-stimulated and nonstimulated cultures in the absence of valinomycin. On day 7 of DEX stimulation, the presence of valinomycin resulted in low p54/52src. Among phosphotyrosine-containing proteins, a 32-34 kDa band, as yet unidentified, showed the most concordant changes with mineralization induction. P32-34 decreased by DEX on days 2 and 8 and increased by low temperature alone or combined with DEX on day 3. On day 7, p32-34 did not change under DEX, but valinomycin selected cells with less phoshpotyrosine-containing p32-34. Taken together, high Src abundance at the start of osteogenic induction followed by a decrease 1 week later is probably related to energy metabolism-dependent induction of mineralization. This is in temporal accord with the increase in Src truncation and fluctuation in mitochondrial membrane potential (which affects mineralization). The reported binding of amino-terminal Src oligopeptide to p32 ADP/ATP carrier in the mitochondrial inner membrane raises the question of its possible involvement in mitochondria-regulated mineralization. J. Cell. Biochem. 69:316-325, 1998. © 1998 Wiley-Liss, Inc.
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  • 22
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    Journal of Cellular Biochemistry 70 (1998), S. 157-158 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
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  • 23
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    Journal of Cellular Biochemistry 70 (1998), S. 159-171 
    ISSN: 0730-2312
    Keywords: nucleus ; nuclear domain ; genome ; nucleolus ; coiled body ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It is becoming clear that the cell nucleus is not only organized in domains but that these domains are also organized relative to each other and to the genome. Specific nuclear domains, enriched in different proteins and RNAs, are often found next to each other and next to specific gene loci. Several lines of investigation suggest that nuclear domains are involved in facilitating or regulating gene expression. The emerging view is that the spatial relationship between different domains and genes on different chromosomes, as found in the nucleolus, is a common organizational principle in the nucleus, to allow an efficient and controlled synthesis and processing of a range of gene transcripts. J. Cell. Biochem. 70:159-171. © 1998 Wiley-Liss, Inc.
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  • 24
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    Journal of Cellular Biochemistry 70 (1998), S. 181-192 
    ISSN: 0730-2312
    Keywords: coiled bodies (CBs) ; gems ; p80 coilin ; RNPs ; RNA processing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Coiled bodies (CBs) are nuclear organelles whose morphology and composition have been conserved from plants to animals. They are highly enriched in components of three different RNA processing pathways. Small nuclear RNAs (snRNAs) involved in pre-mRNA splicing, rRNA processing, and histone mRNA 3′ end maturation all take up residence in CBs. However, CB function(s) remain obscure. This review will focus on recent developments in several aspects of CB structure and function, including exciting new results on their twin organelles, called gems. In particular, the reader will be introduced to a novel hypothesis called the “salmon theory of snRNP biogenesis.” Questions arising from and experiments necessary to test this hypothesis will be discussed. J. Cell. Biochem. 70:181-192, 1998. © 1998 Wiley-Liss, Inc.
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  • 25
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    Journal of Cellular Biochemistry 70 (1998), S. 193-199 
    ISSN: 0730-2312
    Keywords: parathyroid hormone-related peptide ; nucleus ; nucleolus ; intracrine actions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It is becoming increasingly apparent that parathyroid hormone-related peptide (PTHrP) modulates cellular function in a dual mode of action: first, by binding and activating its cognate cell surface G-protein-coupled receptor and, second, by direct intracellular effects following translocation to the nucleus and/or nucleolus of the target cell. Little is presently known about the mechanisms and events that determine the timing and degree of PTHrP nuclear translocation or the role it may serve in normal or dysregulated cellular function. Clarifying the nuclear actions of PTHrP would add significantly to our present understanding of this protein as a signaling molecule during embryonic development and as an oncoprotein whose expression in many tumors correlates with increased tumor aggressiveness and propensity for metastasis. J. Cell. Biochem. 70:193-199, 1998. © 1998 Wiley-Liss, Inc.
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  • 26
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    Journal of Cellular Biochemistry 70 (1998), S. 172-180 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; nuclear structure ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172-180, 1998. © 1998 Wiley-Liss, Inc.
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  • 27
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    Journal of Cellular Biochemistry 69 (1998), S. 81-86 
    ISSN: 0730-2312
    Keywords: cell communication ; osteoblasts ; stromal cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We characterized the formation and regulation of the gap junction in calvarial osteoblasts and in a series of subtypes from marrow stromal cells. The stromal cells included osteogenic, chondro-osteogenic, and endothelial cells. The cell coupling was measured by using fluorescence dye injected into single cells, and its migration to neighboring cells was measured. The functional coupling of cells was highly expressed by the osteoblastic cells. This process is mediated through fast changes in intracellular Ca+2 levels. Calcium ionophore (A 23187) demonstrated an uncoupling effect on the cells. In addition, the exposure of the cells to the parathyroid hormone increased the formation of the gap junction complex; the highest level was demonstrated in the osteoblastic cells. J. Cell. Biochem. 69:81-86, 1998. © 1998 Wiley-Liss, Inc.
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  • 28
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    Journal of Cellular Biochemistry 69 (1998), S. 87-93 
    ISSN: 0730-2312
    Keywords: MAP kinase pathways ; JNK ; human osteoblasts ; interleukin-1β ; UMR-106 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We recently demonstrated the activation of extracellular signal- regulated protein kinase 1 and 2 (ERK1 and ERK2) by IGF-1, FGF-2, and PDGF-BB in normal human osteoblastic (HOB) cells as well as in rat and mouse osteoblastic cells. In this report, we have examined whether c-Jun NH2-Terminal Kinase (JNK) pathway is activated by growth factors and interleukin-1β (IL-1β) in normal HOB and rat UMR-106 cells using immune-complex kinase assay and anti-active JNK antibody, which recognizes activated forms of both JNK1 and JNK2. Results have demonstrated the presence of JNK1 and JNK2 proteins in normal HOB and UMR-106 cells. Both JNK1 and JNK2 were activated by IL-1β. IL-1β preferentially activated JNK pathway in a dose- and time-dependent manner and had little effect on ERK pathway. On the other hand, FGF-2 did not activate JNK but most strongly activated ERK pathway. The activation of JNK was maximal at 20 min whereas maximal activation of ERK1 and ERK2 was observed within 10 min. Results have clearly demonstrated that IL-1β preferentially activates JNK pathway whereas FGF-2 activates ERK pathway in normal human and rat UMR-106 osteoblastic cells. J. Cell. Biochem. 69:87-93, 1998. © 1998 Wiley-Liss, Inc.
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  • 29
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    Journal of Cellular Biochemistry 69 (1998), S. 336-352 
    ISSN: 0730-2312
    Keywords: architectural transcription factor ; nuclear matrix ; osteoblast ; parathyroid hormone ; type I collagen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In connective tissue, cell structure contributes to type I collagen expression. Differences in osteoblast microarchitecture may account for the two distinct cis elements regulating basal expression, in vivo and in vitro, of the rat type I collagen α1(I) polypeptide chain (COL1A1). The COL1A1 promoter conformation may be the penultimate culmination of osteoblast structure. Architectural transcription factors bind to the minor groove of AT-rich DNA and bend it, altering interactions between other trans-acting proteins. Similarly, nuclear matrix (NM) proteins bind to the minor groove of AT-rich matrix-attachment regions, regulating transcription by altering DNA structure. We propose that osteoblast NM architectural transcription factors link cell structure to promoter geometry and COL1A1 transcription. Our objective was to identify potential osteoblast NM architectural transcription factors near the in vitro and in vivo regulatory regions of the rat COL1A1 promoter. Nuclear protein-promoter interactions were analyzed by gel shift analysis and related techniques. NM extracts were derived from rat osteosarcoma cells and from rat bone. The NM protein, NMP4, and a soluble nuclear protein, NP, both bound to two homologous poly(dT) elements within the COL1A1 in vitro regulatory region and proximal to the in vivo regulatory element. These proteins bound within the minor groove and bent the DNA. Parathyroid hormone increased NP/NMP4 binding to both poly(dT) elements and decreased COL1A1 mRNA in the osteosarcoma cells. NP/NMP4-COL1A1 promoter interactions may represent a molecular pathway by which osteoblast structure is coupled to COL1A1 expression. J. Cell. Biochem. 69:336-352. © 1998 Wiley-Liss, Inc.
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  • 30
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    Journal of Cellular Biochemistry 70 (1998), S. 240-251 
    ISSN: 0730-2312
    Keywords: actin ; actin-like proteins ; lamin ; nuclear matrix ; perinuclear actin shells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Perinuclear actin shells have been reported in a variety of organisms. The shells have been identified by staining perinuclear material with fluorescently-labelled phalloidin, but have not been localized to a specific subcellular compartment at the ultrastructural level. We show here that the shells of 3T3 cells lie in the peripheral nuclear matrix. Nuclear shells and matrix actin in other parts of the nucleus are not usually detected by immunohistochemical staining because they are inaccessible to antibodies or to phalloidin. Immunohistochemical detection of nuclear actin is only possible during its deposition at the end of mitosis, or in interphase nuclei that have been extracted with detergent, digested with nucleases and washed with high salt buffers. J. Cell. Biochem. 70:240-251, 1998. © 1998 Wiley-Liss, Inc.
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  • 31
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    Journal of Cellular Biochemistry 68 (1998), S. 247-258 
    ISSN: 0730-2312
    Keywords: SMCs ; bFGF ; collagen fibril structure ; mRNA ; atherosclerotic lesion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular smooth muscle cells (SMCs), the major cellular constituent of an artery, synthesize the bulk of fibrillar collagens, including type V/XI, which regulates heterotypic collagen fibril assembly. Basic fibroblast growth factor (bFGF) is a heparin-binding polypeptide growth factor that has been implicated in important events during the development of atherosclerosis, such as early intimal SMC proliferation. Here we have investigated the effects of bFGF on aortic SMC expression of type V/XI collagen. Treatment of exponentially growing or serum-deprived subconfluent cultures of bovine aortic SMCs with bFGF decreased the steady-state levels of the mRNAs for collagen type V/XI, including α1(V), α2(V), and α1(XI). The effect of bFGF was time dependent with a two- and a fourfold decrease in α2(V) mRNA observed after treatment for 24 and 48 h, respectively. This decrease resulted from a drop in the rate of α2(V) gene transcription; no change was observed in the stability of the α2(V) mRNA. Furthermore, accumulation of collagen protein decreased upon bFGF treatment. As expected, treatment with bFGF increased the rate of proliferation of serum-deprived SMCs, as judged by DNA content in the cultures, thymidine incorporation, and steady-state mRNA levels of the S-phase-expressed histone H3.2. These results suggest that bFGF plays an important role in the regulation of collagen fibril structure, with potential implications for the development and organization of an atherosclerotic lesion. J. Cell. Biochem. 68:247-258, 1998. © 1998 Wiley-Liss, Inc.
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  • 32
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    Journal of Cellular Biochemistry 68 (1998), S. 259-268 
    ISSN: 0730-2312
    Keywords: multifunctional Ca2+/calmodulin-dependent protein kinase ; cardiac isoforms ; muscle differentiation ; cell line Hgc2 ; adult rat heart ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Despite their important role in controlling the cardiac Ca2+ homeostasis, presence and functions of individual isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinase in the heart are not well studied. Here we report on expression of isoforms of the δ class in two differentiation states of the embryonic rat heart-derived cell line H9c2 compared to adult rat heart. Reverse transcription coupled polymerase chain reaction analysis revealed specific expression patterns of four variants of the δ class (δB, δC, δ4, δ9) in adult rat heart, H9c2 myoblasts, and skeletal muscle-like H9c2 myotubes. δC was identified as a common isoform with higher amounts in H9c2 cells and the prominent one in myoblasts. In contrast, expression of δ9 accompanied cardiac as well as skeletal muscle differentiation. Expression of δB, however, was representative for differentiated cardiac muscle, whereas δ4 expression coincided with differentiation into the skeletal muscle-like state. Our results demonstrate differentiation-dependent isoform expression of the δ class of the multifunctional Ca2+/calmodulin-dependent protein kinase of muscle. The identification of cardiac target proteins for this kinase, e.g. the α1-subunit of the L-type Ca2+ channel, the sarcoplasmic reticulum Ca2+-ATPase, phospholamban and the ryanodine receptor define H9c2 myoblasts as a suitable model system for further functional characterization of the identified cardiac δ isoforms. J. Cell. Biochem. 68:259-268, 1998. © 1998 Wiley-Liss, Inc.
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  • 33
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    Journal of Cellular Biochemistry 68 (1998), S. 269-280 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We are using viral oncogene probes to study the pathways by which osteoblast-specific gene expression is induced in ascorbic acid-treated MC3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directly to key cellular regulators and, as a result, represses tissue specific gene expression and blocks differentiation in a wide variety of cell types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family. The p300 family appears to be the primary target for E1A-mediated repression of tissue-specific gene expression in a variety of cell types. We have generated MC3T3-E1 cell lines that stably express either the wild-type 12S product or a mutant that targets p300/CBP, but not the pRB family. Using these constructs to dissect osteoblast differentiation, we found that targeting of p300/CBP appears to be sufficient to repress alkaline phosphatase expression, although a low but functional level of expression can be maintained if the pRB family is not targeted as well. Induction of alkaline phosphatase expression and activity can be dissociated from expression of late-stage markers such as osteocalcin and osteopontin. Surprisingly, cell lines exhibiting severe repression of alkaline phosphatase activity differentiate to a mineral-secreting phenotype much like normal MC3T3-E1 cells. Osteopontin induction is dependent on at least a minimal level of alkaline phosphatase activity, although it is not dependent on induction of alkaline phosphatase at the RNA level. If alkaline phosphatase is supplied exogenously, osteopontin expression can be induced in conditions in which endogenous alkaline phosphatase is severely repressed. J. Cell. Biochem. 68:269-280, 1998. © 1998 Wiley-Liss, Inc.
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  • 34
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    Journal of Cellular Biochemistry 68 (1998), S. 287-297 
    ISSN: 0730-2312
    Keywords: aorta ; mineralization ; calcification ; hydroxyapatite ; inhibitors ; arteriosclerosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na2HPO4 and 0.1 mM CaCl2 were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27-29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis. J. Cell. Biochem. 68:287-297, 1998. © 1998 Wiley-Liss, Inc.
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  • 35
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    Journal of Cellular Biochemistry 68 (1998), S. 281-285 
    ISSN: 0730-2312
    Keywords: QM ; large P-antigen ; 60S ribosomal subunit ; colocalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: QM is a human cDNA originally isolated as a transcript elevated in a nontumorigenic Wilms' tumor microcell hybrid, relative to the tumorigenic parental cell line. The QM gene encodes a 24 kDa basic protein that peripherally associates with the ribosomes. Recently, the gene for this protein has also been shown in Saccharomyces cerevisiaeto encode an essential 60S ribosomal subunit protein that is required for the joining of the 40S and 60S subunits. Since the association of QM with ribosomes can be disrupted with 1M NaCl, which has no effect on the association of core ribosomal proteins, indirect immunofluorescent cell staining was performed to colocalize the QM protein with the human large P-antigen, a core ribosomal protein of the 60S subunit, and to determine whether the assembly of the QM protein onto the 60S ribosomal subunit occurs in the nucleolus or in the cytoplasm. Our results reveal that QM co-localizes with the large P-antigen only to the cytoplasm where the rough endoplasmic reticulum is found and not to the nucleolus where ribosome assembly occurs. This finding suggests that the QM protein is most likely involved in a late step of the 60S subunit assembly and is added to the 60S ribosomal subunit in the cytoplasm and not in the nucleolus. J. Cell. Biochem. 68:281-285, 1998. © 1998 Wiley-Liss, Inc.
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  • 36
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    Journal of Cellular Biochemistry 68 (1998), S. 298-308 
    ISSN: 0730-2312
    Keywords: PEPCK ; adipocytes ; transcription ; fatty acids ; fibrates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Phosphoenolpyruvate carboxykinase (PEPCK) exerts a glyceroneogenic function in adipocytes in which transcription of its gene is increased by unsaturated fatty acids and fibrates. We used cultured rat adipose tissue fragments and 3T3-F442A adipocytes to show that the antidiabetic thiazolidinedione BRL 49653, a ligand and an activator of the γ isoform of peroxisome proliferator activated receptors (PPARγ), is a potent inducer of PEPCK mRNA. In 3T3-F442A adipocytes, the effect of BRL 49653 is rapid and concentration dependent, with a maximum reached at 1 μM and a half-maximum at 10-100 nM. PEPCK mRNA is similarly induced by the natural ligand of PPARγ, the 15-deoxy-Δ12-14 prostaglandin J2. These observations strongly suggest that PPARγ is a primary regulator of PEPCK gene expression in adipocytes. Dexamethasone at 10 nM repress induction of PEPCK mRNA by 1 μM BRL 49653, 0.32 mM oleate, or 1 mM clofibrate, in a cycloheximide-independent manner. The antiglucocorticoid RU 38486 prevents dexamethasone action, demonstrating involvement of the glucocorticoid receptor. Stable transfectants of 3T3-F442A adipocytes bearing -2100 to +69 base pairs of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene respond to 1 μM BRL 49653 or 1 mM clofibrate by a large increase in CAT activity, which is prevented by the simultaneous addition of 10 nM dexamethasone. Hence, in adipocytes, glucocorticoids act directly through the 5′-flanking region of the PEPCK gene to repress, in a dominant fashion, the stimulation of PEPCK gene transcription by thiazolidinediones and fibrates. J. Cell. Biochem. 68:298-308, 1998. © 1998 Wiley-Liss, Inc.
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  • 37
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    Journal of Cellular Biochemistry 68 (1998), S. 309-327 
    ISSN: 0730-2312
    Keywords: in vitro replication ; ors8 ; Oct-1 transcription factor ; POU domain ; mammalian autonomously replicating DNA sequence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A 186-base pair fragment of ors8, a mammalian autonomously replicating DNA sequence isolated by extrusion of nascent monkey DNA in early S phase, has previously been identified as the minimal sequence required for replication function in vitro and in vivo. This 186-base pair fragment contains, among other sequence characteristics, an imperfect consensus binding site for the ubiquitous transcription factor Oct-1. We have investigated the role of Oct-1 protein in the in vitro replication of this mammalian origin. Depletion of the endogenous Oct-1 protein, by inclusion of an oligonucleotide comprising the Oct-1 binding site, inhibited the in vitro replication of p186 to approximately 15-20% of the control, whereas a mutated Oct-1 and a nonspecific oligonucleotide had no effect. Furthermore, immunodepletion of the Oct-1 protein from the HeLa cell extracts by addition of an anti-POU antibody to the in vitro replication reactioninhibited p186 replication to 25% of control levels. This inhibition of replication could be partially reversed to 50-65% of control levels, a two- to threefold increase, upon the addition of exogenous Oct-1 POU domain protein.Site-directed mutagenesis of the octamer binding site in p186 resulted in a mutant clone, p186-MutOct, which abolished Oct-1 binding but was still able to replicate as efficiently as the wild-type p186. The results suggest that Oct-1 protein is an enhancing component in the in vitro replication of p186 but that its effect on replication is not caused through direct binding to the octamer motif. J. Cell. Biochem. 68:309-327, 1998. © 1998 Wiley-Liss, Inc.
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  • 38
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    Journal of Cellular Biochemistry 68 (1998), S. 328-338 
    ISSN: 0730-2312
    Keywords: cell proliferation ; tumor progression ; EGF receptor ; ErbB ; HER1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an activating ligand for the EGF receptor (HER1/ErbB1) and the high-affinity receptor for diphtheria toxin (DT) in its transmembrane form (proHB-EGF). HB-EGF was immunolocalized within human benign and malignant prostatic tissues, using monospecific antibodies directed against the mature protein and against the cytoplasmic domain of proHB-EGF. Prostate carcinoma cells, normal glandular epithelial cells, undifferentiated fibroblasts, and inflammatory cells were not decorated by the anti-HB-EGF antibodies; however, interstitial and vascular smooth muscle cells were highly reactive, indicating that the smooth muscle compartments are the major sites of synthesis and localization of HB-EGF within the prostate. In marked contrast to prostatic epithelium, proHB-EGF was immunolocalized to seminal vesicle epithelium, indicating differential regulation of HB-EGF synthesis within various epithelia of the reproductive tract. HB-EGF was not overexpressed in this series of cancer tissues, in comparison to the benign tissues. In experiments with LNCaP human prostate carcinoma cells, HB-EGF was similar in potency to epidermal growth factor (EGF) in stimulating cell growth. Exogenous HB-EGF and EGF each activated HER1 and HER3 receptor tyrosine kinases and induced tyrosine phosphorylation of cellular proteins to a similar extent. LNCaP cells expressed detectable but low levels of HB-EGF mRNA; however, proHB-EGF was detected at the cell surface indirectly by demonstration of specific sensitivity to DT. HB-EGF is the first HER1 ligand to be identified predominantly as a smooth muscle cell product in the human prostate. Further, the observation that HB-EGF is similar to EGF in mitogenic potency for human prostate carcinoma cells suggests that it may be one of the hypothesized stromal mediators of prostate cancer growth. J. Cell. Biochem. 68:328-338, 1998. © 1998 Wiley-Liss, Inc.
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  • 39
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    Journal of Cellular Biochemistry 68 (1998), S. 366-377 
    ISSN: 0730-2312
    Keywords: PC-1 ; insulin action ; insulin resistance ; insulin receptor ; tyrosine kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: An elevated content of membrane glycoprotein PC-1 has been observed in cells and tissues of insulin resistant patients. In addition, in vitro overexpression of PC-1 in cultured cells induces insulin resistance associated with diminished insulin receptor tyrosine kinase activity. We now find that PC-1 overexpression also influences insulin receptor signaling at a step downstream of insulin receptor tyrosine kinase, independent of insulin receptor tyrosine kinase. In the present studies, we employed Chinese hamster ovary cells that overexpress the human insulin receptor (CHO IR cells; ∼106 receptors per cell), and transfected them with human PC-1 c-DNA (CHO IR PC-1). In CHO IR PC-1 cells, insulin receptor tyrosine kinase activity was unchanged, following insulin treatment of cells. However, several biological effects of insulin, including glucose and amino acid uptake, were decreased. In CHO IR PC-1 cells, insulin stimulation of mitogen-activated protein (MAP) kinase activity was normal, suggesting that PC-1 overexpression did not affect insulin receptor activation of Ras, which is upstream of MAP kinase. Also, insulin-stimulated phosphatidylinositol (PI)-3-kinase activity was normal, suggesting that PC-1 overexpression did not interfere with the activation of this enzyme by insulin receptor substrate-1. In these cells, however, insulin stimulation of p70 ribosomal S6 kinase activity was diminished. These studies suggest, therefore, that, in addition to blocking insulin receptor tyrosine kinase activation, PC-1 can also block insulin receptor signaling at a post-receptor site. J. Cell. Biochem. 68:366-377, 1998. © 1998 Wiley-Liss, Inc.
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  • 40
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    Journal of Cellular Biochemistry 68 (1998), S. 355-365 
    ISSN: 0730-2312
    Keywords: mechanical loading ; gene expression ; osteopontin ; myeloperoxidase ; rats ; differential display ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function. J. Cell. Biochem. 68:355-365, 1998. © 1998 Wiley-Liss, Inc.
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  • 41
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    Journal of Cellular Biochemistry 68 (1998), S. 339-354 
    ISSN: 0730-2312
    Keywords: glutathione ; reactive oxygen intermediates ; HIV ; signal transduction ; cytokines ; redox state ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α). While virtually every cell responds to TNF-α with gene activation, the extent of TNF-α-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-α membrane interaction, are part of this TNF-α-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-α. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-α challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV- normal fibroblasts. In contrast, following TNF-α challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-α-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-α challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-α, suggest a biochemical rationale for the recognized TNF-α AIDS-KS clinical correlation. J. Cell. Biochem. 68:339-354, 1998. © 1998 Wiley-Liss, Inc.
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  • 42
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    Journal of Cellular Biochemistry 70 (1998), S. 468-477 
    ISSN: 0730-2312
    Keywords: scleraxis ; transcription factor ; FGF ; chondrocyte ; bHLH ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Scleraxis is a basic helix-loop-helix-type transcription factor that is expressed in sclerotome. Fibroblast growth factor (FGF) is one of the cytokines produced by the cells in skeletal tissues and is a potent modulator of skeletogenesis. The aim of this study was to examine the effects of FGF on the expression of scleraxis in chondrocyte-like cells, TC6. In these cells, scleraxis mRNA was constitutively expressed as a 1.2kb message at a high level in contrast to its low levels of expression in fibroblast-like cells or osteoblast-like cells. Upon treatment with FGF, scleraxis mRNA level was decreased within 12 h. This effect was at its nadir at 24 h and the scleraxis mRNA level returned to its base line level by 48 h. The FGF effect was maximal at 1 ng/ml. FGF effects on scleraxis were blocked by actinomycin D but not by cycloheximide, suggesting the involvement of transcriptional events that do not require new protein synthesis. The FGF effects on scleraxis were blocked by genistein, suggesting the involvement of tyrosine kinase in the post-receptor signaling. TGFβ treatment of TC6 cells enhanced scleraxis mRNA expression; however, combination of the saturation doses of FGF and TGFβ resulted in suppression of scleraxis mRNA level. BMP2 also suppressed scleraxis mRNA expression in TC6 cells and no further suppression was observed in combination with FGF. These results indicate that scleraxis is expressed in chondrocyte-like TC6 cells and it is one of the targets of FGF action in these cells. J. Cell. Biochem. 70:468-477. © 1998 Wiley-Liss, Inc.
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  • 43
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    Journal of Cellular Biochemistry 70 (1998), S. 489-506 
    ISSN: 0730-2312
    Keywords: hematopoiesis ; protein interaction ; EMSA ; nucleolin ; nucleophosmin/NPM/B23 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The human myeloid nuclear differentiation antigen, MNDA, is expressed only in myelomonocytic and a subset of B lymphoid hematopoietic cells. MNDA is uniformly distributed throughout the interphase cell nucleus and associates with chromatin, but does not bind specific DNA sequences. We recently demonstrated that MNDA binds nucleolin and nucleophosmin/NPM/B23 and both of these nuclear proteins bind the ubiquitous zinc finger transcription factor YY1. Investigations of the possible effect of MNDA on the interaction between YY1 and NPM, showed that MNDA bound YY1 directly under both in vitro and in vivo conditions. The MNDA-YY1 interaction enhanced the affinity of YY1 for its target DNA and decreased its rate of dissociation. The N-terminal half (200 amino acids) of MNDA was sufficient for maximum enhancement of YY1 DNA binding and a portion of this sequence was responsible for binding YY1. MNDA participated in a ternary complex with YY1 and the YY1 target DNA element. The results show that MNDA affects the ability of YY1 to bind its target DNA sequnce and that MNDA participates in a ternary complex possibly acting as a cofactor to impart lineage specific features to YY1 function. J. Cell. Biochem. 70:489-506, 1998. © 1998 Wiley-Liss, Inc.
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  • 44
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    Journal of Cellular Biochemistry 70 (1998), S. 507-516 
    ISSN: 0730-2312
    Keywords: type X collagen; transcription ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Collagen X is expressed specifically in hypertrophic chondrocytes within cartilage that is undergoing endochondral ossification. The chicken collagen X gene is transcriptionally regulated, and under the control of multiple cis elements within the distal promoter region (-4,442 to -558 base pairs from the transcription start) as well as the proximal region (-558 to +1). Our previous data (LuValle et al., [1993] J. Cell Biol. 121:1173-1179) demonstrated that the proximal sequence directed high reporter gene activity in the three cell types tested (hypertrophic chondrocytes, immature chondrocytes, and fibroblasts), while distal elements acted in an additive manner to repress the effects of the proximal sequence on reporter gene activity in non-collagen X expressing cells only (immature chondrocytes and fibroblasts). We show here that elements within the proximal sequence (nucleotides -557 to -513) are necessary for the cell-specific expression of type X collagen by hypertrophic chondrocytes. These elements bind to proteins of 100 kDa in all three cell types, and 47 kDa in non-collagen X expressing cells. Reporter gene activity in hypertrophic chondrocytes is reduced to the levels seen in non-collagen X-expressing cells in the absence of these elements. J. Cell. Biochem. 70:507-516, 1998. © 1998 Wiley-Liss, Inc.
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  • 45
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    Journal of Cellular Biochemistry 70 (1998), S. 478-488 
    ISSN: 0730-2312
    Keywords: osteoporosis ; osteopenia ; aging ; bone formation ; growth factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: One of the universal characteristics of the long bones and spines of middle-age and older mammals is a loss in bone mass (osteopenia). In humans, if this bone loss is severe enough, it results in osteoporosis, a skeletal disorder characterized by a markedly increased incidence of fractures with sequelae that may include pain, loss of mobility, and in the event of hip fracture, even death within a relatively few months of injury. An important contributing factor to the development of osteopororsis appears to be a diminution in the number and activity of osteoblasts responsible for synthesizing new bone matrix. The findings in the present and other similar studies suggest that this reduction in osteoblast number and activity is due to an age-related diminution in the size and osteogenic potential of the bone marrow osteoblast progenitor cell (OPC or CFU-f) compartment. We previously postulated that these regressive changes in the OPC/CFU-f compartment occurred in old animals because of a reduction in the amount and/or activity of TGF-β1, an autocrine growth factor important in the promotion of OPC/CFU-f proliferation and differentiation. In support of this hypothesis, we now report that (1) the osteogenic capacity of the bone marrow of 24-month-old BALB/c mice, as assessed in vivo, is markedly reduced relative to that of 3-4-month-old animals, (2) that the matrix of the long bones of old mice contains significantly less TGF-β than that of young mice, (3) that OPC's/CFU-f's isolated from old mice produce less TGF-β in vitro than those recovered from young mice, and (4) that OPC's/CFU-f's from old mice express significantly more TGF-β receptor (Types I, II, and III) than those of young animals and that such cells are more responsive in vitro to exogenous recombinant TGF-β1. We also find that colony number and proliferative activity of OPC's/CFU-f's of young mice and old mice, respectively, are significantly reduced when incubated in the presence of neutralizing TGF-β1 antibody. Collectively, these data are consistent with the hypothesis that in old male mice the reduction in the synthesis and, perhaps, availability from the bone matrix of TGF-β1 contributes to a diminution in the size and development potential of the bone marrow osteoprogenitor pool. J. Cell. Biochem. 70:478-488. © 1998 Wiley-Liss, Inc.
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  • 46
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    Journal of Cellular Biochemistry 70 (1998), S. 517-527 
    ISSN: 0730-2312
    Keywords: arthritis ; cartilage ; gene regulation ; kinases ; signaling ; tissue inhibitors of metalloproteinases ; transforming growth factor beta ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn-over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF-β), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP-3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF-β-mediated induction of TIMP-3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased TIMP-3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP-3 gene to TGF-β. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF-β induction of TIMP-3. H7 and genistein also suppressed TGF-β-induced TIMP-3 protein expression. These results suggest that TGF-β signaling for TIMP-3 gene induction involves H7-sensitive serine/threonine kinase as well as herbimycin A- and genistein-sensitive protein tyrosine kinases. J. Cell. Biochem. 70:517-527, 1998. © 1998 Wiley-Liss, Inc.
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  • 47
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    Journal of Cellular Biochemistry 71 (1998), S. 243-253 
    ISSN: 0730-2312
    Keywords: T-lymphocyte ; apoptosis ; signal transduction ; HgCl2 ; tyrosine phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Exposure to Hg2+ at a wide range of concentrations (approximately 1-100 μM) more or less caused the death of murine thymic T-lymphocytes, and exposure to 1 μM but not 10 μM (or more) of Hg2+ induced DNA fragmentation. Exposure of cells to Hg2+ caused phosphorylation of multiple cellular proteins at the tyrosine residue in a concentration-dependent manner. We found that not only the DNA fragmentation induced by 1 μM Hg2+ but also the cell death bypassing DNA fragmentation caused by 10 μM or more Hg2+ was partly inhibited by protein kinase inhibitors such as staurosporine and herbimycin A. This result suggested the involvement of a protein phosphorylation-linked signal in the mechanism of the Hg2+-mediated cell death with or without DNA fragmentation. Analysis of proteins by both one- and two-dimensional electrophoresis and immunoblot showed that a 52-kDa Shc protein was heavily phosphorylated by an early signal delivered by a high concentration of Hg2+, which also phosphorylated extracellular signal-regulated kinase 1 (ERK1; p44) and ERK2 (p42) of the mitogen-activated protein kinase (MAPK) family in a concentration- and time-dependent manner. The c-Jun amino terminal kinase (p54), which is a distant relative of the MAPK family, was also phosphorylated by the treatment with Hg2+. This eventually formed the signaling cascade that ended with a nuclear target by phosphorylating c-jun at the serine 73. This phosphorylation of c-jun was inhibited by staurosporine. These results suggest that a high level of Hg2+-mediated protein phosphorylation-linked signal induces rapid cell death bypassing DNA fragmentation, whereas a lower level induces cell death accompanying DNA fragmentation. This conclusion in turn implies that DNA fragmentation is not always a prerequisite for the signal transduction-dependent cell death of T-lymphocytes. J. Cell. Biochem. 71:243-253, 1998. © 1998 Wiley-Liss, Inc.
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  • 48
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    Journal of Cellular Biochemistry 71 (1998), S. 254-263 
    ISSN: 0730-2312
    Keywords: lysyl oxidase ; cyclooxygenase-1 ; type I collagen α1 ; prostaglandin E2 ; prostaglandin E2 receptors ; cyclic AMP ; interleukin-1β ; transforming growth factor-β ; forskolin ; 11-deoxy PGE1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a recent communication, we demonstrated that prostaglandin E2 (PGE2) lowers basal while it ablates interleukin-1β( (IL-1β) and transforming growth factor-β (TGFβ) upregulated lysyl oxidase (LO) mRNA levels. Correspondingly, PGE2 increases cyclooxygenase-1 (COX1) mRNA in diploid, human embryo lung fibroblasts (IMR90) [Roy et al., 1996]. We now report that these actions by PGE2 are routed through cAMP via the PGE2, EP2 receptor. Among the PGE2 receptor types, the IMR90 predominantly express the EP2 mRNA. These cells also express EP3 and EP4 mRNA at comparatively low levels. Northern blot analyses show that 11-deoxy PGE1, an EP2/EP4 agonist, emulates the action of PGE2. In a similar manner to PGE2, 11-deoxy PGE1 decreases basal and TGF-β induced type I collagen α1 (COL) mRNA, basal and IL-1β induced LO mRNA while it increases COX1 mRNA. Sulprostone, an EP3/EP1 agonist, has no effect on the expression of these three genes. Forskolin, an adenylate cyclase activator, acts in a very similar manner to PGE2or 11-deoxy PGE1. It suppresses both basal and TGF-β induced COL mRNA levels. Both PGE2 and 11-deoxy PGE1 increase cAMP to a level comparable with forskolin. The role of the EP2 receptor in controlling collagen production is further underscored in the immortalized Rat-1 fibroblasts, derived from Fischer rat embryos, which do not express detectable EP2 mRNA. In these cells, PGE2 has little effect on COL mRNA level, whereas forskolin increases it. Furthermore, forskolin increases cAMP level in Rat-1 cells, whereas PGE2 does not. Overall, these results illustrate that much of the PGE2 action on the expression of COL, LO, and COX1 genes is mediated through the EP2 receptor and a subsequent increase in intracellular cAMP. J. Cell. Biochem. 71:254-263, 1998. © 1998 Wiley-Liss, Inc.
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  • 49
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    Journal of Cellular Biochemistry 71 (1998), S. 286-301 
    ISSN: 0730-2312
    Keywords: heart ; development ; MAPK ; MEK ; MEKK ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The loss of ability to proliferate (terminal differentiation) and reduction in capability to resist ischemia are key phenomena observed during postnatal development of the heart. Mitogen-activated protein kinases (MAPKs) mediate signaling pathways for cell proliferation/differentiation and stress responses such as ischemia. In this study, the expression of these kinases and their associated kinases were investigated in rat heart ventricle. Extracts of 1-, 10-, 20-, 50-, and 365-day-old rat heart ventricles were probed with specific antibodies and their immunoreactivities were quantified by densitometry. Most of the mitogenic protein kinases including Raf1, RafB, Mek1, Erk2, and Rsk1 were significantly down-regulated, whereas the stress signaling kinases, such as Mlk3, Mekk1, Sek1, Mkk3, and Mapkapk2 were up-regulated in expression during postnatal development. Most MAP kinases including Erk1, JNKs, p38 Hog, as well as Rsk2, however, did not exhibit postnatal changes in expression. The proto-oncogene-encoded kinases Mos and Cot/Tpl 2 were up-regulated up to two- and four-fold, respectively, during development. Pak1, which may be involved in the regulation of cytoskeleton as well as in stress signaling, was downregulated with age, but the Pak2 isoform increased only after 50 days. All of these proteins, except RafB, were also detected in the isolated adult ventricular myocytes at comparable levels to those found in adult ventricle. Tissue distribution studies revealed that most of the protein kinases that were up-regulated during heart development tended to be preferentially expressed in heart, whereas the downregulated protein kinases were generally expressed in heart at relatively lesser amounts than in most of other tissues. J. Cell. Biochem. 71:286-301, 1998. © 1998 Wiley-Liss, Inc.
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  • 50
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 71 (1998), S. 313-327 
    ISSN: 0730-2312
    Keywords: articular cartilage repair ; tissue engineering ; collagen type II ; collagen type IX ; collagen network ; pyridinium crosslinks ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of collagen types II and IX into a three-dimensional organized collagen network that can balance the swelling pressure of the proteoglycan/ water gel. To determine whether cartilage engineered in vitro contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6 weeks and analyzed with respect to the composition and ultrastructure of collagen by using biochemical and immunochemical methods and scanning electron microscopy. Total collagen content and the concentration of pyridinium crosslinks were significantly (57% and 70%, respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However, the fractions of collagen types II, IX, and X and the collagen network organization, density, and fibril diameter in engineered cartilage were not significantly different from those in natural articular cartilage. The implications of these findings for the field of tissue engineering are that differentiated chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a tissue similar to natural articular cartilage on an ultrastructural scale. J. Cell. Biochem. 71:313-327, 1998. © 1998 Wiley-Liss, Inc.
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  • 51
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 71 (1998), S. 340-350 
    ISSN: 0730-2312
    Keywords: vitamin D analogues ; vitamin D receptor ; ligand binding ; limited protease digestion ; ligand-dependent gel shift assay ; gene regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear hormone 1α,25-dihydroxyvitamin D3 (VD) has important cell-regulatory functions but also a strong calcemic effect. Therefore, various VD analogues have been synthesized and screened for their biological profile. In order to gain more insight into the molecular basis of the high antiproliferative but low calcemic action of the VD analogue EB1089, we characterized this compound in comparison to five structurally related VD analogues. The activities of the six VD analogues in in vitro assays (limited protease digestion assays for determining interaction with monomeric vitamin D receptor (VDR), ligand-dependent gel shift assays for showing the increase of DNA binding of VDR-retinoid X receptor (RXR) heterodimers, and reporter gene assays on different types of VD response elements for demonstrating the efficacy in nuclear VD signalling) were found to represent their biological potency (antiproliferative effect on different malignant cell lines). In this series, EB1089 proved to be the most potent VD analogue; that is, every structural modification (20-epi configuration, cis-configuration at position C24, or changes at the ethyl groups at position C25) appeared to reduce the determined activities mediated through the VDR of these analogues. Moreover, the modifications of EB1089 resulted in a loss of VD response element selectivity, suggesting that this parameter is very critical for the biological profile of this VD analogue. J. Cell. Biochem. 71:340-350, 1998. © 1998 Wiley-Liss, Inc.
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  • 52
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 71 (1998), S. 351-362 
    ISSN: 0730-2312
    Keywords: IGFBP ; cAMP ; PKA ; prostaglandin ; bone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNA and accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low Mr bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP. J. Cell. Biochem. 71:351-362, 1998. © 1998 Wiley-Liss, Inc.
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  • 53
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 71 (1998), S. 21-35 
    ISSN: 0730-2312
    Keywords: heat shock protein ; heat shock genes ; heat shock element ; heat shock factor ; basal transcription elements ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Exposure of 9L rat brain tumor cells to 40-100 μM CdCl2 for 2 h leads to an induction of a wide spectrum of heat shock proteins (HSPs). We have demonstrated that induction of the 70-kDa HSP (HSP70) and enhanced expression of its cognate (HSC70) by cadmium are concentration dependent and that the induction kinetics of these HSP70s are different. The increased synthesis of the HSP70s is accompanied by the increase in hsp70 and hsc70 mRNA levels, indicative of transcriptional regulation of the heat shock genes. Electrophoretic mobility shift assay (EMSA) using probes encompassing heat shock element (HSE), TATA, GC, and CCAAT boxes derived from the promoter regions of the heat shock genes shows distinguished binding patterns between hsp70 and hsc70 genes in both control and cadmium-treated cells. The results indicate that, in addition to the HSEs, the basal transcription elements are important in the regulation of the heat shock genes. The binding patterns of the corresponding transcription factors of these elements are examined by EMSA by using extended promoter fragments from respective heat shock genes with sequential addition of excess oligonucleotides encompassing individual transcription elements. Taken together, our results show that the differential induction of hsp70 and hsc70 involves multiple transcription factors that interact with HSE, TATA, GC, and CCAAT boxes. J. Cell. Biochem. 71:21-35, 1998. © 1998 Wiley-Liss, Inc.
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