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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 68 (1998), S. 1-7 
    ISSN: 0730-2312
    Keywords: cell stress ; heat shock ; σ32 ; magnetic fields ; ribonuclease protection assay ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The mechanism of interaction between weak electromagnetic fields and cells is not understood. As a result, the health effect(s) induced by exposure to these fields remains unclear. In addition to questions relating to the site of initial magnetic field (MF) interactions, the nature of the cell's response to these perturbations is also unclear. We examined the hypothesis that the cells respond to MFs in a manner similar to other environmental stressors such as heat. Using the bacterium Escherichia coli, we examined the mRNA levels of σ32, a protein that interacts with RNA polymerase to help it recognize a variety of stress promoters in the cell. Our data show that the intracellular level of σ32 mRNA is enhanced following a 15-min exposure to a 60 Hz, 1.1 mT magnetic field. J. Cell. Biochem. 68:1-7, 1998. © 1998 Wiley-Liss, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 22-30 
    ISSN: 0730-2312
    Keywords: cathepsin-B ; tissue transglutaminase ; mesangial cell apoptosis ; mRNA expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mesangial matrix is a dynamic structure which modulates mesangial cell function. Since accumulation of matrix precedes the development of focal glomerulosclerosis, we studied the effect of different matrices on mesangial cell (MC) apoptosis. Suspended mesangial cells became apoptotic in a time dependent manner. Collagen type III did not modulate MC apoptosis when compared to cells grown on plastic. MCs grown on Matrigel, collagen type I and IV showed an increased number of apoptotic cells when compared to MCs grown on plastic. DNA end-labeling further confirmed these observations. MCs grown on Matrigel showed enhanced (P 〈 0.05) mRNA expression for tissue transglutaminase (TTG) and cathepsin-B. Mesangial cells grown on Matrigel also showed enhanced expression of superoxide dismutase (SOD). We conclude that mesangial cells require attachment to the matrix for their survival and alteration of the quality of matrix modulates mesangial cell apoptosis. J. Cell. Biochem. 68:22-30, 1998. © 1998 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 50-61 
    ISSN: 0730-2312
    Keywords: Sp1 ; p62 ; interaction assay ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The transcription factor Sp1 plays an important role in the expression of many cellular genes. In studies of proteins that associate with Sp1, a 62-kDa glycoprotein was found in immunoprecipitates of Sp1. This protein was detected in these immunoprecipitates by the monoclonal antibody, RL2, which was originally raised against nuclear pore proteins but was subsequently found to recognize an epitope that contains O-linked N-acetylglucosamine (O-GlcNAc). The association of this protein with Sp1 could be blocked by SDS denaturation of the protein complex. Western blot analysis of the Sp1 immunoprecipitate using antibodies to p62 nucleoporin indicated that this nuclear pore protein associates with Sp1. Furthermore, immunoprecipitation of p62 nucleoporin resulted in the coprecipitation of Sp1. Recombinant p62, expressed as a GST-fusion protein using a vaccinia virus system, also interacted with both recombinant and native Sp1. This interaction between p62 and Sp1 required the C-terminus of p62 and the C-terminus was able to bind Sp1, albeit less efficiently than native p62. A mammalian two-hybrid interaction assay was devised in which p62 was fused to the Gal4 DNA-binding domain. This system also indicated that p62, through its C-terminus, interacts with Sp1 in the living cell. We propose that this interaction of a nuclear pore protein with Sp1 may reflect the nuclear organization required to bring transcribable DNA in contact with the transcription factors. J. Cell. Biochem. 68:50-61, 1998. © 1998 Wiley-Liss, Inc.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 378-388 
    ISSN: 0730-2312
    Keywords: apoptosis ; growth suppression ; retinoic acid receptors ; ovarian cancer ; AHPN ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have used conformationally restricted retinoids to investigate the role of individual RAR subtypes and RXR in mediating the growth response of ovarian tumor cells to retinoids. Our results show that treatment of all-trans-RA-sensitive CAOV-3 cells with retinoids that bind and activate a single RAR or RXR led to a partial inhibition of growth. Treatment of all-trans-RA- resistant SKOV-3 cells did not alter growth. Maximum inhibition of growth, comparable to that observed following treatment with natural retinoids such as all-trans-RA and 9-cis-RA, was obtained only following treatment with a combination of an RAR-selective compound and an RXR-selective one. These results suggest that activation of both RAR and RXR classes is required in order to obtain maximum inhibition of ovarian tumor cell growth by retinoids. In addition, one compound, AHPN, was found to inhibit both RA-sensitive CAOV-3 and RA-resistant SKOV-3 cells. Further study of the effects of this retinoid showed that AHPN acts through an apoptotic pathway. Taken together, our results suggest that retinoids may serve as effective anti-proliferative agents in the treatment of ovarian cancer. J. Cell. Biochem. 68:378-388, 1998. © 1998 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 83-89 
    ISSN: 0730-2312
    Keywords: pH ; osteoblasts ; collagen synthesis ; alkaline phosphatase activity ; glycolysis ; DNA synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of medium pH on the activity of cultured human osteoblasts was investigated in this study. Osteoblasts derived from explants of human trabecular bone were grown to confluence and subcultured. The first-pass cells were incubated in Hepes-buffered media at initial pHs adjusted from 7.0 to 7.8. Osteoblast function was evaluated by measuring lactate production, alkaline phosphatase activity, proline hydroxylation, DNA content, and thymidine incorporation. Changes in medium pH were determined from media pHs recorded at the beginning and end of the final 48 h incubation period. As medium pH increased through pH 7.6, collagen synthesis, alkaline phosphatase activity, and thymidine incorporation increased. DNA content increased from pH 7.0 to 7.2, plateaued from pH 7.2 to 7.6, and increased again from pH 7.6 to 7.8. The changes in the medium pH were greatest at pHs 7.0 and 7.8, modest at pHs 7.4 and 7.6, and did not change at 7.2, suggesting that the pHs are migrating towards pH 7.2. Lactate production increased at pH 7.0 but remained constant from 7.2 to 7.8. These results suggest that in the pH range from 7.0-7.6 the activity of human osteoblasts increases with increasing pH, that this increase in activity does not require an increase in glycolytic activity, and that pH 7.2 may be the optimal pH for these cells. J. Cell. Biochem. 68:83-89, 1998. © 1998 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 181-188 
    ISSN: 0730-2312
    Keywords: magnetic fields ; HSP70 gene expression ; human HSP70 promoter ; c-myc protein binding sites ; cellular stress ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated c-myc protein-binding sites on the HSP70 promoter as modulators of the induction of HSP70 gene expression in response to magnetic field stimulation (8μT at 60Hz) and whether the presence of c-myc protein potentiates transactivation of HSP70 expression. A 320 base pair region in the HSP70 promoter (+1 to -320) was analyzed. This region contains two c-myc-protein binding sites with consensus sequences located at -230 and -160 nucleotide positions (relative to the transcription initiation site) and overlapping with the region reported for the regulation of HSP70 gene expression by c-myc protein. This promoter region is upstream of other regulatory sequences, including the heat shock element (HSE), AP-2, and serum response element (SRE). Transfectants containing both c-myc protein-binding sites, HSP-MYC A and HSP-MYC B, and exposed to magnetic fields showed a 3.0-fold increase in expression of CAT activity as compared with sham-exposed control transfectants. Transfectants containing one c-myc binding site, HSP-MYC A, and exposed to magnetic fields showed a 2.3-fold increase in CAT expression. Transfectants in which both HSP-MYC A and HSP-MYC B binding sites were deleted showed no magnetic field sensitivity; values were virtually identical with sham-exposed controls. If the c-myc expression vector was not co-transfected with the constructs containing myc-binding sites, there was no difference in the expression of CAT activity between magnetically stimulated and sham-exposed controls, although both responded to heat shock. These data suggest that endogenous elevated levels of myc protein contribute to the induction of HSP70 in response to magnetic field stimulation. J. Cell. Biochem. 69:181-188, 1998. © 1998 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 446-456 
    ISSN: 0730-2312
    Keywords: IGF-I ; IGF-II ; cAMP ; PKA ; PKC ; prostaglandin ; osteoblasts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone cells synthesize and respond to IGF-I and IGF-II which contribute to bone remodeling and linear growth. In osteoblasts, prostaglandin (PG)E2 stimulates IGF-I but not IGF-II synthesis through a cAMP-dependent protein kinase A (PKA)-related event. However, protein kinase C (PKC) activation by PGE2 enhances replication and protein synthesis by less differentiated periosteal cells more so than in osteoblast-enriched cultures from fetal rat bone. Using various PGs and other PKA and PKC pathway activators, the importance of these aspects of PGE2 activity has now been examined on IGF receptors in these bone cell culture models. PGE2 and other agents that activate PKA enhanced 125I-IGF-II binding to type 2 IGF receptors on both cell populations. In contrast, agents that activate PKC enhanced 125I-IGF-I binding to type 1 receptors on less differentiated bone cells, and of these, only phorbol myristate acetate (PMA), which activates PKC in a receptor-independent way, was effective in osteoblast-enriched cultures. No stimulator increased total type 1 receptor protein in either cell population. Consequently, ligand binding to type 1 and type 2 IGF receptors is differentially modulated by specific intracellular pathways in bone cells. Importantly, changes in apparent type 1 receptor number occur rapidly and may do so at least in part through post-translational effects. These results may help to predict new ways to manipulate autocrine or paracrine actions by IGFs in skeletal tissue. J. Cell. Biochem. 68:446-456, 1998. © 1998 Wiley-Liss, Inc.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 436-445 
    ISSN: 0730-2312
    Keywords: mouse ; PDI family proteins ; retinoic acid ; dibutyryl cAMP ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be co-immunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation. J. Cell. Biochem. 68:436-445, 1998. © 1998 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 427-435 
    ISSN: 0730-2312
    Keywords: α2-macroglobulin ; albumin ; placenta ; zinc ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have investigated the binding and internalization of α2-macroglobulin and serum albumin by human placental syncytiotrophoblast cells in vitro. The time course (obtained at 4°C) of α2-macroglobulin binding indicated that an equilibrium was reached after 4 h. The binding of 125I-labelled α2-macroglobulin to syncytiotrophoblast cells was competitively reduced in the presence of excess unlabelled α2-macroglobulin. When the concentration-dependence of binding was examined over a wide concentration range, non-linear regression analysis yielded a Kd of 6.4 nM. In the case of albumin, binding was weak and ligand dissociated from the cell surface during aqueous washing making it impractical to analyze the binding reaction. In other experiments, syncytiotrophoblast cells were incubated with 125I-labelled α2-macroglobulin at 37°C. Under these conditions, trypsin-resistant cell-associated radioactivity increased with time consistent with ligand internalization. 125I-Labelled-ligand was internalized with a t1/2 of about 5 min. After a lag period some radioactivity was released back into the incubation medium. When measured at times up to 210 min, this was found to consist of mostly TCA-precipitable material that had been lost from the cell surface. However, when the incubation was extended to 24 h, almost 15% of the initial cell-associated radioactivity was released to the extracellular medium as TCA-soluble material, consistent with a slow rate of ligand degradation. The specific binding of 65Zn-labelled α2M was similar to that of the 125I-labelled ligand and trypsin-resistance measurements provided evidence of α2M-mediated 65Zn uptake. These results support a role for syncytiotrophoblast in the metabolism of α2-macroglobulin during pregnancy and are also consistent with a role for α2-macroglobulin in the maternal-fetal transport of zinc. J. Cell. Biochem. 68:427-435, 1998. © 1998 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 457-471 
    ISSN: 0730-2312
    Keywords: coated vesicles ; acetylcholine receptors ; AP180 ; myotube ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Monoclonal antibodies were generated to vesicular membranes of clathrin coated vesicles enriched for acetylcholinesterase (AChE). One of these, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunoblots of muscle extracts and brain purified clathrin coated vesicles show that C172 recognizes a 100 kd band in muscle, but a 180 kd band in brain. Western blots of purified AP180 protein stained with the two antibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to known sequences of AP180 were used to map the epitope for C172 within the brain AP180 sequence. On immunoblots of digested AP180, all AP180 antibodies and C172 recognized a 100 kd tryptic fragment, however only C172 recognized a smaller 60 kd. Our results suggest that the C172 epitope is located within amino acids 305-598 of the AP180 sequence. Confocal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distinct from that observed when cells are stained with γ adaptin antibody which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quantitative analysis of C172 reactive vesicles revealed a significant increase in number of vesicles present around the nuclei associated with the acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with homology to the neuron-specific coated vesicle protein AP180, is present in muscle cells associated with vesicles showing significant concentration around postsynaptic nuclei present in close proximity to AChR clusters. J. Cell. Biochem. 68:457-471, 1998. © 1998 Wiley-Liss, Inc.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 472-483 
    ISSN: 0730-2312
    Keywords: taxol ; microtubules ; vimentin ; intermediate filaments ; protein phosphorylation ; protein kinases ; inhibitors ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Taxol, a microtubule stabilizing agent, has been extensively investigated for its antitumor activity. The cytotoxic effect of taxol is generally attributed to its antimicrotubule activity and is believed to be cell cycle dependent. Herein, we report that taxol induces hyperphosphorylation and reorganization of the vimentin intermediate filament in 9L rat brain tumor cells, in concentration- and time-dependent manner. Phosphorylation of vimentin was maximum at 10-6 M of taxol treatment for 8 h and diminished at higher (10-5 M) concentration. Enhanced phosphorylation of vimentin was detectable at 2 h treatment with 10-6 M taxol and was maximum after 12 h of treatment. Taxol-induced phosphorylation of vimentin was largely abolished in cells pretreated with staurosporine and bisindolymaleimide but was unaffected by H-89, KT-5926, SB203580, genistein, and olomoucine. Thus, protein kinase C may be involved in this process. Hyperphosphorylation of vimentin was accompanied by rounding up of cells as revealed by scanning electron microscopy. Moreover, there was a concomitant reorganization of the vimentin intermediate filament in the taxol-treated cells, whereas the microtubules and the actin microfilaments were less affected. Taken together, our data demonstrate that taxol induces hyperphosphorylation of vimentin with concomitant reorganization of the vimentin intermediate filament and that this process may be mediated via a protein kinase C signaling pathway. J. Cell Biochem. 68:472-483, 1998. © 1998 Wiley-Liss, Inc.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 511-524 
    ISSN: 0730-2312
    Keywords: actin ; permeability ; reoxygenation ; signal transduction ; cytoskeletal rearrangement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Hypoxia/reoxygenation injury to cultured endothelial cells results in cytoskeletal rearrangement and second messenger activation related to increased monolayer junctional permeability. Cytoskeletal rearrangement by reactive oxygen species may be related to specific activation of the phospholipase D (PLD) pathway. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 μM) or metabolites of the PLD pathway for 1-60 min. Changes in cAMP levels, Ca2+ levels, PIP2 production, filamin distribution, and intercellular gap formation are then quantitated. H2O2-induced filamin translocation from the membrane to the cytosol occurs after 1-min H2O2 treatment, while intercellular gap formation significantly increases after 15 min. H2O2 and phosphatidic acid exposure rapidly decrease intracellular cAMP levels, while increasing PIP2 levels in a Ca2+-independent manner. H2O2-induced cAMP decreases are prevented by inhibiting phospholipase D. H2O2-induced cytoskeletal changes are prevented by inhibiting phospholipase D, phosphatidylinositol-4-phosphate kinase, phosphoinositide turnover, or by adding a synthetic peptide that binds PIP2. These data indicate that metabolites produced downstream of H2O2-induced PLD activation may mediate filamin redistribution and F-actin rearrangement. J. Cell. Biochem. 68:511-524, 1998. © 1998 Wiley-Liss, Inc.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 1-12 
    ISSN: 0730-2312
    Keywords: two-hybrid system ; vitamin D receptor ; retinoid X receptor ; vitamin D ; protein L7 ; basic region leucine zipper domain ; coregulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes. Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D. Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR. Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937. Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast. We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element. Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid. Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR. Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements. J. Cell. Biochem. 69:1-12, 1998. © 1998 Wiley-Liss, Inc.
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  • 14
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 484-499 
    ISSN: 0730-2312
    Keywords: YY1 ; zinc finger ; high-molecular-weight complex ; plasmid transfection ; nuclear matrix association ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: YY1 is a zinc finger-containing transcription factor that can both repress and activate transcription. YY1 appears to use multiple mechanisms to carry out its diverse functions. Recently, it was observed that YY1 can exist in multiple nuclear compartments. In addition to being present in the nuclear extract fraction, YY1 is also a component of the nuclear matrix. We show that YY1 can be sequestered in vivo into a high-molecular-weight complex and can be dislodged from this complex either by treatment with formamide or by incubation with an oligonucleotide containing the YY1 DNA binding site sequence. By transfecting plasmids expressing various YY1 deletion constructs and subsequent nuclear fractionation, we have identified sequences necessary for association with the nuclear matrix. These sequences (residues 256-340) co-localized with those necessary for in vivo sequestration of YY1 into the high-molecular-weight complex. We have also characterized YY1 sequences necessary for repression of activated transcription (residues 333-371) and those necessary for masking of the YY1 transactivation domain (residues 371-397). Sequences that repress activated transcription partially overlap YY1 sequences necessary for association with the nuclear matrix. However, these sequences are distinct from those that appear to mask the YY1 transactivation domain. The potential role of nuclear matrix association in controlling YY1 function is discussed. J. Cell. Biochem. 68:484-499, 1998. © 1998 Wiley-Liss, Inc.
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  • 15
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 500-510 
    ISSN: 0730-2312
    Keywords: transcription factor ; nuclear matrix ; YY1 ; amino acids ; functional regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The multifunctional transcription factor YY1 is associated with the nuclear matrix. In osteoblasts, the interaction of several nuclear matrix-associated transcription factors with the bone specific osteocalcin gene contributes to tissue-specific and steroid hormone-mediated transcription. A canonical nuclear matrix targeting signal (NMTS) is present in all members of the AML/CBFβ transcription factor family, but not in other transcription factors. Therefore, we defined sequences that direct YY1 (414 amino acids) to the nuclear matrix. A series of epitope tagged deletion constructs were expressed in HeLa S3 and in human Saos-2 osteosarcoma cells. Subcellular distribution was determined in whole cells and nuclear matrices in situ by immunofluorescence. We demonstrated that amino acids 257-341 in the C-terminal domain of YY1 are necessary for nuclear matrix association. We also observed that sequences within the N-terminal domain of YY1 permit weak nuclear matrix binding. Our data further suggest that the Gal4 epitope tag contains sequences that affect subcellular localization, but not targeting to the nuclear matrix. The targeted association of YY1 with the nuclear matrix provides an additional level of functional regulation for this transcription factor that can exhibit positive and negative control. J. Cell. Biochem. 68:500-510, 1998. © 1998 Wiley-Liss, Inc.
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  • 16
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 13-18 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; replication origin ; topoisomerase II-mediated DNA loop excision ; DNA loop anchorage sites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions. J. Cell. Biochem. 69:13-18, 1998. © 1998 Wiley-Liss, Inc.
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  • 17
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 19-29 
    ISSN: 0730-2312
    Keywords: interleukin-1 ; reactive oxygen species ; nitric oxide ; c-fos ; collagenase ; chondrocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interleukin-1β (IL-1) is implicated in cartilage destruction in arthritis through promotion of matrix metalloproteinase production. Upregulation of collagenase gene expression by IL-1 is known to require the transactivators Fos and Jun. Recently, reactive oxygen species (ROS) have been suggested to act as intracellular signaling molecules mediating the biological effects of cytokines. Here, we demonstrated ROS production by IL-1-stimulated bovine chondrocytes and that neutralizing ROS activity by the potent antioxidant, N-acetylcysteine, or inhibiting endogenous ROS production by diphenyleneiodonium (DPI), significantly attenuated IL-1-induced c-fos and collagenase gene expression. The inhibitory effect of DPI implicates enzymes such as NADPH oxidase in the endogenous production of ROS. Chondrocytes were also found to produce nitric oxide (NO) upon IL-1 stimulation. That NO may mediate part of the inducing effects of IL-1 was supported by the observation that L-NG-monomethylarginine, a NO synthase inhibitor, partially inhibited IL-1-regulated collagenase expression. Moreover, treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, was sufficient to induce collagenase mRNA levels. In summary, our results suggest that ROS released in response to IL-1 may function as second messengers transducing extracellular stimuli to their targets in the nucleus, leading to augmentation of gene expression. J. Cell. Biochem. 69:19-29, 1998. © 1998 Wiley-Liss, Inc.
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  • 18
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 55-62 
    ISSN: 0730-2312
    Keywords: TGF-α ; antisense oligonucleotides ; head and neck cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interruption of an autocrine growth pathway involving TGF-α and EGFR may inhibit tumor growth and improve survival in head and neck cancer patients. We previously demonstrated that biopsy specimens and established cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress TGF-α and its receptor, epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies showed that TGF-α and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting a role for this ligand-receptor pair in an autocrine growth pathway. To confirm that TGF-α contributes to autocrine growth, we examined the effect of down regulation of TGF-α protein on SCCHN cell proliferation. Treatment of 6 SCCHN cell lines with antisense oligodeoxynucleotides targeting the translation start site of human TGF-α mRNA decreased TGF-α protein production by up to 93% and reduced cell proliferation by a mean of 76.2% compared to a 9.7% reduction with sense oligonucleotide (range P〈0R 〉 = 0.036-0.0001). TGF-α antisense oligonucleotide exposure also decreased TGF-α protein levels in normal oropharyngeal mucosal epithelial cells, however their growth rate was not affected. These findings indicate that TGF-α is participating in an autocrine signaling pathway in transformed, but not in normal mucosal epithelial cells, that promotes proliferation. J. Cell. Biochem. 69:55-62, 1998. © 1998 Wiley-Liss, Inc.
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  • 19
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    Journal of Cellular Biochemistry 69 (1998), S. 30-43 
    ISSN: 0730-2312
    Keywords: hyperthermia ; calreticulin ; chaperone complexes ; prompt glycosylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Acute heat stress leads to the glycosylation of a “prompt” stress glycoprotein, P-SG67/64, identified as calreticulin. In the present study, we used immunoprecipitation to investigate the interactions of P-SG/calreticulin with other proteins during cellular recovery from heat stress. In heat-stressed CHO and M21 cells, both glycosylated and unglycosylated P-SGs interact with HSP90, GRP94, GRP78, and the other prompt stress glycoprotein, P-SG50, in an ATP-independent manner. Specificity of HSP-P-SG interactions was determined by chemical cross-linking with the homo-bifunctional agent DSP (3,3′-dithiobis[succinimidyl propionate]). Characterization of the cross-linked complexes involving calreticulin and heat shock proteins (HSPs) showed an average mass of 400-600 kDa by gel filtration chromatography. Overall, the consistent association of glycosylated and unglycosylated calreticulin with P-SG50 and unglycosylated HSPs suggests that P-SG/calreticulin is an active member of the cast of glycone/aglycone chaperones that cooperate to achieve cellular recovery from acute heat stress. J. Cell. Biochem. 69:30-43, 1998. © 1998 Wiley-Liss, Inc.
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  • 20
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    Journal of Cellular Biochemistry 69 (1998), S. 63-71 
    ISSN: 0730-2312
    Keywords: angiotensin II ; G proteins ; Src tyrosine kinases ; c-Fos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Angiotensin II stimulates a biphasic activation of Raf-1, MEK, and ERK in WB liver epithelial cells. The first peak of activity is rapid and transient and is followed by a sustained phase. Angiotensin II also causes a rapid activation of p21ras in these cells. Moreover, two Src family kinases (Fyn and Yes) were activated by angiotensin II in a time- and concentration-dependent manner. Microinjection of antibodies against Fyn and Yes blocked angiotensin II-induced DNA synthesis and c-Fos expression in WB cells, indicating an obligatory involvement of these tyrosine kinases in the activation of the ERK cascade by angiotensin II. Finally, substantial reduction of the angiotensin II-stimulated activation of Fyn, Raf-1, ERK, and expression of c-Fos by pertussis toxin pretreatment argues that G proteins of the Gi family as well as the Gq family are involved in angiotensin II-mediated mitogenic pathways in WB cells. J. Cell. Biochem. 69:63-71, 1998. © 1998 Wiley-Liss, Inc.
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  • 21
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    Journal of Cellular Biochemistry 69 (1998), S. 44-54 
    ISSN: 0730-2312
    Keywords: genistein ; breast cancer ; p21WAF1/CIP1 ; G2/M arrest ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Genistein has been proposed to be responsible for lowering the rate of breast cancer in Asian women but the mechanism for this chemopreventive effect in vivo is unknown. In this study, we present in vitro evidence that genistein inhibits cell proliferation similarly in ER-positive and ER-negative human breast carcinoma cell lines. This inhibition is associated with specific G2/M arrest and induction of p21WAF1/CIP1 expression. Genistein results in a five- to six-fold increase in p21WAF1/CIP1 mRNA levels and a three- to four-fold increase in protein levels, only a 1.5-fold increase in p21WAF1/CIP1 transcription but a three- to six-fold increase in p21WAF1/CIP1 mRNA stability. The increase in p21WAF1/CIP1 is followed by increased apoptosis. The similar effects of genistein on a number of breast carcinoma cell lines with different ER and p53 status suggest that the actions of genistein reported here are mediated through ER and p53 independent mechanisms. The chemopreventive effects of genistein in vivo could be mediated along an identical or similar anti-proliferative pathway. J. Cell. Biochem. 69:44-54, 1998. © 1998 Wiley-Liss, Inc.
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  • 22
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    Journal of Cellular Biochemistry 69 (1998), S. 72-80 
    ISSN: 0730-2312
    Keywords: human oocytes ; immunogold labeling ; splicing factors ; coilin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The distribution of two splicing components (snRNP and SC-35) and coilin were studied by immunogold/electron microscopy in human oocytes from antral follicles at different levels of transcriptional activity (i.e., active, intermediate, and inactive). The results showed a decrease of snRNPs and SC-35 in the karyoplasm as the oocytes progress from a transcriptionally active to the inactive state. The main areas of accumulation of both these splicing components in all stages of oocytes appeared to be the interchromatin granule clusters (IGCs). Within the IGCs, the two splicing components seemed to be spatially segregated, with the snRNPs predominantly bound to the fibrillar region, whereas the SC-35 factors are being enriched in the granular zone. The p80 coilin was found only in the nucleolus-like body (NLB), which is present in all three stages of oocytes; no coiled bodies were evident. These data are consistent with the notion that splicing occurs in the karyoplasm and that the splicing components are mobilized from a storage site (IGCs) to the site of action. J. Cell. Biochem. 69:72-80, 1998. © 1998 Wiley-Liss, Inc.
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  • 23
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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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  • 24
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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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  • 25
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    Journal of Cellular Biochemistry 69 (1998), S. 104-116 
    ISSN: 0730-2312
    Keywords: mRNA export ; cell cycle ; gene transfection ; cultured mammalian cells ; hnRNP L ; nuclear transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human β-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless β-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes. J. Cell. Biochem. 69:104-116, 1998. © 1998 Wiley-Liss, Inc.
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  • 26
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    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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  • 27
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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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  • 28
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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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  • 29
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    Journal of Cellular Biochemistry 69 (1998), S. 453-462 
    ISSN: 0730-2312
    Keywords: Rous sarcoma virus ; chondrocytes ; matrix calcification ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification. J. Cell. Biochem. 69:453-462, 1998. © 1998 Wiley-Liss, Inc.
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  • 30
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    Journal of Cellular Biochemistry 69 (1998), S. 436-452 
    ISSN: 0730-2312
    Keywords: small heat shock proteins ; TNFα ; phosphorylation mutant ; SB203580 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The role of murine Hsp25 phosphorylation in the protection mediated by this protein against TNFα- or H2O2-mediated cytotoxicity was investigated in L929 cell lines expressing wild type (wt-) or nonphosphorylatable (mt-) Hsp25. We show that mt-Hsp25, in which the phosphorylation sites, serines 15 and 86, were replaced by alanines, is still efficient in decreasing intracellular reactive oxygen species levels and in raising glutathione cellular content, leading the protective activity of mt-Hsp25 against oxidative stress to be identical to that of wt-Hsp25. To independently investigate the role of Hsp25 phosphorylation, we blocked TNFα-induced phosphorylation of wt-Hsp25 using SB203580, a specific inhibitor of the P38 MAP kinase. This treatment did not abolish the protective activity of Hsp25 against TNFα. The pattern of Hsp25 oligomerization was also analyzed, showing mt-Hsp25 to constitutively display large native sizes, as does wt-Hsp25 after TNFα treatment in the presence of SB203580. Our results, therefore, are consistent with the possibility that the hyperaggregated form of Hsp25 is responsible for the protective activity against oxidative stress and that the phosphorylation of serines 15 and/or 86 by interfering with this structural reorganization, may lead to the inactivation of Hsp25 protective activity. J. Cell. Biochem. 69:436-452, 1998. © 1998 Wiley-Liss, Inc.
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  • 31
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    Journal of Cellular Biochemistry 70 (1998), S. 604-615 
    ISSN: 0730-2312
    Keywords: DNA replication ; apoptosis ; DNA cleavage ; endonuclease ; Bal 31 ; topological domains ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal 31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5 - 1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.J. Cell. Biochem. 70:604-615, 1998. © 1998 Wiley-Liss, Inc. © 1998 Wiley-Liss, Inc.
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  • 32
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    Journal of Cellular Biochemistry 70 (1998), S. 596-603 
    ISSN: 0730-2312
    Keywords: poly(ADP-ribose) ; PARP ; nuclear matrix ; noncovalent interactions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system. J. Cell. Biochem. 70:596-603. © 1998 Wiley-Liss, Inc.
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  • 33
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    Journal of Cellular Biochemistry 71 (1998), S. 11-20 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; DNA replication ; α-polymerase ; confocal microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G1 phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18-22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [3H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11-20, 1998. © 1998 Wiley-Liss, Inc.
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  • 34
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    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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  • 35
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    Journal of Cellular Biochemistry 70 (1998), S. 8-21 
    ISSN: 0730-2312
    Keywords: activin A ; bone marrow stromal cells ; gene regulation ; promoter activity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Activin A, a member of the TGF-β superfamily, plays roles in differentiation and development, including hematopoiesis. Our previous studies indicated that the expression of activin A by human bone marrow cells and monocytes is highly regulated by inflammatory cytokines and glucocorticoids. The present study was undertaken to investigate the regulation of activin A gene expression in the human bone marrow stromal cell lines L87/4 and HS-5, as well as in primary stromal cells. Northern blots demonstrated that, like primary stromal cells, the cell lines expressed four activin A RNA transcripts (6.4, 4.0, 2.8, and 1.6 kb), although distribution of the RNA among the four sizes varied. The locations of the 5′ ends of the RNAs were investigated by Northern blots and RNase protection assays. The results identified a transcription start site at 212 nucleotides upstream of the translation start codon. In addition, luciferase expression assays of a series of deletion constructs were used to identify regulatory sequences upstream of the activin A gene. A 58 bp upstream sequence exhibits promoter activity. However, severalfold higher expression requires a positive element consisting of an additional 71 bp of the upstream region. Promoter activity was also identified between 2.5 and 3.6 kb upstream of the start codon. These findings suggest that expression of activin A at the transcriptional level follows complex patterns of regulation. J. Cell. Biochem. 70:8-21, 1998. © 1998 Wiley-Liss, Inc.
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  • 36
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    Journal of Cellular Biochemistry 69 (1998), S. 506-521 
    ISSN: 0730-2312
    Keywords: heart ; development ; CaMPK ; cAPK ; CDK ; cGPK ; Kkialre ; PKC ; Wee1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: During early postnatal development, cardiomyocytes, which comprise about 80% of ventricular mass and volume, become phenotypically developed to facilitate their contractile functions and terminally differentiated to grow only in size but not in cell number. These changes are due to the expression of contractile proteins as well as the regulation of intracellular signal transduction proteins. In this study, the expression patterns of several protein kinases involved in various cardiac functions and cell-cycle control were analyzed by Western blotting of ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats. The expression level of cAMP-dependent protein kinase was slightly decreased (20%) over the first year, whereas no change was detected in cGMP-dependent protein kinase I. Calmodulin-dependent protein kinase II, which is involved in Ca2+ uptake into the sarcoplasmic reticulum, was increased as much as ten-fold. To the contrary, the expressions of protein kinase C-α and ι declined 77% with age. Cyclin-dependent protein kinases (CDKs) such as CDK1, CDK2, CDK4, and CDK5, which are required for cell-cycle progression, abruptly declined to almost undetectable levels after 10-20 days of age. In contrast, other CDK-related kinases, such as CDK8 or Kkialre, did not change significantly or increased up to 50% with age, respectively. Protein kinases implicated in CDK regulation such as CDK7 and Wee1 were either slightly increased in expression or did not change significantly. All of the proteins that were detected in ventricular extracts were also identified in isolated cardiac myocytes in equivalent amounts and analyzed for their relative expression in ten other adult rat tissues. J. Cell. Biochem. 69:506-521, 1998. © 1998 Wiley-Liss, Inc.
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  • 37
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    Journal of Cellular Biochemistry 70 (1998), S. 22-28 
    ISSN: 0730-2312
    Keywords: Msx-1 ; Msx-2 ; homeobox ; adhesion ; sorting ; cadherin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Msx-1 and Msx-2 are two closely related homeobox genes expressed in cephalic neural crest tooth buds, the optic cup endocardial cushions, and the developing limb [Hill and Davidson, 1991; Monaghan et al., 1991; Robert et al., 1991]. These sites correspond to regions of active cell segregation and proliferation under the influence of epithelial-mesenchymal cell interactions [Brown et al., 1993; Davidson et al., 1991], suggesting that Msx-1 and Msx-2 regulate cell-cell interactions. We have investigated the potential relationship between expression of the Msh homeobox genes (Msx-1 and Msx-2) and cadherin-mediated cell adhesion and cell sorting. We report that cell lines stably expressing Msx-1 or Msx-2 differentially sort on the basis of Msh gene expression. We demonstrate in vitro that initial cell aggregation involves calcium-dependent adhesion molecules (cadherins) and that Msh genes regulate cadherin-mediated adhesion. These results support the hypothesis that Msh genes play a role in the regulation of cell-cell adhesion and provide a link between the genetic phenomena of homeobox gene expression and cellular events involved in morphogenesis, including cell sorting and proliferation. J. Cell. Biochem. 70:22-28, 1998. © 1998 Wiley-Liss, Inc.
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  • 38
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    Journal of Cellular Biochemistry 70 (1998), S. 29-37 
    ISSN: 0730-2312
    Keywords: small GTPase ; membrane traffic ; vesicles ; transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Eukaryotic cells achieve complexity by compartmentalizing a subset of cellular functions into membrane-bound organelles. Maintaining this high level of cellular organization requires precise regulation of traffic between membranes. This task is accomplished, in part, by rab proteins. How these small GTPases regulate membrane traffic between cellular compartments is not clear. Here we report the characterization of a novel rab GTPase from the soil amoebae Dictyostelium discoideum. The predicted coding sequence of the new rab gene, Dictyostelium rab11b, encodes a protein of 25 kD containing all the structural hallmarks of a rab GTPase. Comparison of the sequence with the GenBank database and cladistic analysis demonstrated Dictyostelium rab11b to be a divergent member of the rab11 branch of rab proteins. Southern analysis revealed the presence of related genes in Dictyostelium. RNAse protection assays showed the Dictyostelium rab11b gene to be expressed at uniform levels throughout growth and development. Gene deletion experiments revealed that Dictyostelium rab11b was not essential for growth or development. Conceivably, the function of rab11b may be redundant with that of related genes in this organism. J. Cell. Biochem. 70:29-37, 1998. © 1998 Wiley-Liss, inc.
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  • 39
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    Journal of Cellular Biochemistry 71 (1998), S. 74-81 
    ISSN: 0730-2312
    Keywords: prostaglandin ; phospholipase A2 ; age ; tumor necrosis factor-α ; transforming growth factor-β1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The eicosanoids, including prostaglandin E2 (PGE2) and other bioactive arachidonic acid metabolites, are important local mediators of bone remodeling. Presumably, the limited or excessive synthesis of the eicosanoids could compromise bone homeostasis. We have noted that the stimulated release of arachidonic acid by adult male donor derived human osteoblast-like (hOB) cells exceeded the stimulated release measured for female-derived hOB cells by 1.5-fold. Assays of PGE2 biosynthesis by cytokine-stimulated hOB cells also demonstrated a sex-linked difference, such that male hOB cell PGE2 production exceeded female cell production by 1.6-2.2-fold. The calcium-dependent cytoplasmic phospholipase A2 activity in subcellular fractions prepared from hOB cell homogenates was higher in both the cytosolic (1.6-fold) and particulate (1.5-fold) fractions from the male cells than in those prepared from female hOB cells, suggesting a molecular basis for the observed sexually dimorphic characteristics related to arachidonic acid metabolism by hOB cells. The relatively limited capacity of the female cells may limit needed intracellular and intercellular signaling during bone remodeling, thereby contributing to the development of bone pathology. J. Cell. Biochem. 71:74-81, 1998. © 1998 Wiley-Liss, Inc.
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  • 40
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    Journal of Cellular Biochemistry 71 (1998), S. 63-73 
    ISSN: 0730-2312
    Keywords: integrin ; activation epitopes ; ligand binding ; focal adhesions ; cytoplasmic domains ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The anti-integrin β1 MAb 15/7 sometimes may be a reporter of integrin activation or ligand occupancy. However, certain β1 tail deletions eliminate ligand binding despite inducing maximal constitutive 15/7 expression [Puzon-Mclaughlin et al. (1996): J Biol Chem 271:16580-16585]. Here we describe β1 tail mutations (e.g., double point mutations [D759L/F763L, F766L/E769L], or replacement of the β1 tail by the β5 tail) that prevent rather than induce constitutive appearance of the 15/7 epitope. Despite variable losses of constitutive 15/7 epitope, these mutants all retained a similar inducible 15/7 epitope component as seen upon incubation with GRGDSP peptide ligand. In addition, constitutive 15/7 expression did not correlate with integrin localization into focal adhesions. In conclusion, we show for the first time for a fully functional integrin that specific mutations within the β1 tail can down-regulate the constitutive appearance of an extracellular conformation defined by MAb 15/7. Because this regulation occurs away from the ligand binding site and does not correlate with responsiveness to integrin ligand, cell adhesion, or localization into focal adhesions, a novel type of conformational regulation is suggested. J. Cell. Biochem. 71:63-73, 1998. © 1998 Wiley-Liss, Inc.
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  • 41
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    Journal of Cellular Biochemistry 71 (1998), S. 82-95 
    ISSN: 0730-2312
    Keywords: M-line proteins ; titin ; expression ; antibody perturbation ; immunocytochemistry ; cardiomyocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A rat polyclonal anti-M-line protein antiserum and three mouse monoclonal anti-titin antibodies (E2, F3, and A12) were used to study the spatiotemporal relationship between M-line proteins and titin during myofibril assembly in cultured chicken cardiomyocytes by immunofluorescence microscopy. In day 2 cultures, M-line proteins and titin were detected as punctate staining in most cardiomyocytes, which possessed many nonstriated fibrils. At a late stage (day 3 cultures), M-line proteins were incorporated into dot-like structures along nonstriated fibrils, while titin staining was continuous on these structures. As development progressed, M-line proteins were registered in periodic pattern in the mid-A band. In cardiomyocytes from day 5 cultures, the titin bands were separated by an unstained region, and achieved their adult doublet pattern. Thus, the organization of titin in the sarcomere appears to occur later than that of M-line proteins in the M-line. Our morphological data indicate that the early registration of M-line proteins in primitive myofibrils may guide titin filament alignment via interaction between M-line proteins and titin. In order to investigate the role of M-line proteins in the assembly of titin filaments, anti-M-line protein or anti-titin antibodies were introduced into cultured cardiomyocytes by electroporation to functionally bind the respective proteins, and the profile of myofibril assembly was examined. Cardiomyocytes from day 2-3 cultures with incorporated anti-M-line protein antibodies became shrunk, and exhibited defective myofibrillar assembly, as shown by the failure of titin to assemble into a typical sarcomeric pattern. Incorporation of anti-titin antibody E2, which recognizes the M-line end domain of titin, resulted in the failure of M-line proteins organized into the M-line structure, as shown by random, sporadic staining with anti-M-line protein antibody. These studies confirm the essential role of M-line proteins in the organization of titin filaments in the sarcomere and that the interaction between titin and M-line proteins is crucial to the formation of the M-line structure. J. Cell. Biochem. 71:82-95, 1998. © 1998 Wiley-Liss, Inc.
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  • 42
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    Journal of Cellular Biochemistry 71 (1998), S. 109-115 
    ISSN: 0730-2312
    Keywords: fluid shear stress ; adrenomedullin ; endothelial cell ; SSRE ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular endothelial cells are potent modulators of vascular tone in response to shear stress. Levels of vasoactive peptides such as adrenomedullin (AM), endothelin-1 (ET-1), C-type natriuretic peptide (CNP), and nitric oxide (NO) are affected by fluid shear stress. AM, a potent vasodilator and suppressor of smooth muscle cell proliferation, contains the shear stress responsive element (SSRE) “GAGACC” in its promoter region. To examine the role of AM in the shear stress response, cultured human aortic endothelial cells (HAoECs) were exposed to fluid shear stresses of 12 and 24 dynes/cm2 in a cone-plate shear stress loading apparatus for various time periods, and the levels of AM gene expression and peptide secretion from HAoECs were measured by Northern blotting analysis and radioimmunoassay (RIA), respectively. Both AM gene transcription and AM peptide levels were down-regulated by fluid shear stress in a time- and magnitude-dependent manner. Our results demonstrate that the normal level of arterial shear stress down-regulates AM expression in HAoECs, suggesting that AM participates in the modulation of vascular tone by fluid shear stress. J. Cell. Biochem. 71:109-115, 1998. © 1998 Wiley-Liss, Inc.
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  • 43
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    Journal of Cellular Biochemistry 71 (1998), S. 96-108 
    ISSN: 0730-2312
    Keywords: androgens ; androgen receptor ; antiandrogens ; differentiation ; osteoblasts ; proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (∼4,000/nucleus) of androgen receptors (AR). Treatment with 5α-dihydrotestosterone (5α-DHT) inhibited the proliferation of hFOB/AR-6 cells in a dose-dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (〈200/nucleus). In hFOB/AR-6 cells, co-treatment with the specific AR antagonist, hydroxyflutamide abolished 5α-DHT-induced growth inhibition. Steady-state levels of transforming growth factor-β1 (TGF-β1) and TGF-β-induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR-6 cells with 5α-DHT, suggesting a role for the TGF-β1-TIEG pathway in mediating 5α-DHT-induced growth inhibition of hFOB/AR-6 cells. In support of this, co-treatment of hFOB/AR-6 cells with TGF-β1 (40 pg/ml) reversed the 5α-DHT-induced growth inhibition, whereas TGF-β1 alone at this dose had no effect on hFOB/AR-6 cell proliferation. Furthermore, treatment of hFOB/AR-6 cells with 5α-DHT and testosterone (10-8 M) inhibited basal and 1,25-(OH)2D3-induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast. J. Cell. Biochem. 71:96-108, 1998. © 1998 Wiley-Liss, Inc.
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  • 44
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    Journal of Cellular Biochemistry 71 (1998), S. 127-139 
    ISSN: 0730-2312
    Keywords: GAPDH gene expression ; spermatogenesis ; meiotic and postmeiotic cells ; heat shock ; polyadenylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in addition to being a classic glycolytic enzyme, is a multifunctional protein involved in relevant cell functions such as DNA replication, DNA repair, translational control of gene expression, and apoptosis. Although the multifunctional nature of GAPDH suggests versatility in the mechanisms regulating its expression, no major qualitative changes and few quantitative changes in the GAPDH transcripts have been reported. While studying the expression of GAPDH during spermatogenesis, we detected alternative initiations to TATA box and alternative splicings in the 5′ region of the pre-mRNA, resulting in at least six different types of mRNAs. The amount and the polyadenylation of the GAPDH transcripts increased in mature testis in relation to immature testis and further increased when cell suspensions from mature testis were exposed to heat shock. These results suggest that alternative initiation, alternative splicing, and polyadenylation could provide the necessary versatility to the regulation of the expression of this multifunctional protein during spermatogenesis. J. Cell. Biochem. 71:127-139, 1998. © 1998 Wiley-Liss, Inc.
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  • 45
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    Journal of Cellular Biochemistry 71 (1998), S. 140-148 
    ISSN: 0730-2312
    Keywords: calmodulin ; calcineurin ; protein phosphatase ; calcium-binding protein ; regucalcin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The regulatory effect of regucalcin on Ca2+/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca2+ (100 μM) and calmodulin (0.30 μM). Thess increases were clearly inhibited by the addition of regucalcin (0.50-1.0 μM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 μg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 μM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 μM) in the presence of Ca2+ (1 and 10 μM). This increase was completely inhibited by the presence of regucalcin (0.12 μM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 μM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca2+/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin. J. Cell. Biochem. 71:140-148, 1998. © 1998 Wiley-Liss, Inc.
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  • 46
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    Journal of Cellular Biochemistry 71 (1998), S. 116-126 
    ISSN: 0730-2312
    Keywords: alkaline phosphatase ; osteogenic induction ; pp60Src ; tyrosine phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cyclosporin A (CsA) induces osteoporosis but not through direct activation of osteoclasts. CsA also inhibits cell-mediated mineralization in marrow stromal cell culture, whereas the tyrphostin AG-1478 increases mineralization. These antagonistic effects on mineralization were used to discern molecules that underwent phosphorylation changes in association with their opposing effects on mineralization. In parallel, quantitative changes in Src protein were followed. Multiple dexamethasone (DEX)-stimulated stromal cell cultures were grown with and without a mineralization-inhibiting dose (0.1 μM) of CsA and were harvested on different days of DEX stimulation. Immunoblots of gel-fractionated cell extracts showed that the most noticeable changes in tyrosine phosphorylated proteins (TPP) were seen on day 8 of DEX stimulation. At least 15 TPP bands, mostly smaller than 53 kDa, were more prominent in CsA-treated cultures on day 8. Under CsA, Src protein quantity decreased on day 8, but its cleavage product (52/54 kDa) was sixfold more abundant then on day 7. Day 8 was chosen to test the effect of AG-1478 on the CsA-induced TPP changes. Dimethyl sulfoxide (DMSO) alone, the solvent of AG-1478, increased mineralization in CsA-treated versus CsA-untreated cultures and slightly decreased Src and its cleavage product. AG-1478 at 5 μM, in CsA cultures increased the specific alkaline phosphatase activity threefold, with a slight change in mineralization relative to controls grown with DMSO alone. This was accompanied by decreased intensity of several TPP bands smaller than 36 kDa. In contrast, treatment with 50 μM of AG-1478 increased the intensity of TPP bands at the same molecular size range. This high AG-1478 dose decreased cell counts selecting mineralizing cells. The results indicate that increased Src protein cleavage product on day 8 by CsA is associated with mineralization inhibition, which is opposed by DMSO and 50-μM AG-1478, thus antagonizing the effect of CsA on mineralization. Direct or indirect interaction between Src and TPP, antagonistically affected by CsA and AG-1478, is likely to underlay cellular control of mineralization. Changes in p19 and p29 intensity showed association with mineralization that was reflected by a significant direct and inverse correlation, respectively, with calcium precipitation per cell. J. Cell. Biochem. 71:116-126, 1998. © 1998 Wiley-Liss, Inc.
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  • 47
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    Journal of Cellular Biochemistry 71 (1998), S. 158-168 
    ISSN: 0730-2312
    Keywords: glycosylation ; lysosomal targeting ; lysozyme ; monensin ; myeloperoxidase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The role of the N-terminal sequence of myeloperoxidase in the intracellular targeting was examined by using glycosylated lysozyme as a reporter. A fusion protein was constructed in which the presequence residues -18 through -6 of the lysozyme moiety had been replaced by residues 1-158 of prepromyeloperoxidase. Expression of the fusion protein in Chinese hamster ovary cells demonstrated its partial secretion and partial intracellular retention. The latter was accompanied by trimming the myeloperoxidase prosequence off the lysozyme moiety. The rate of the retention of the lysozyme fusion protein was higher than that of glycosylated lysozyme that had been expressed in cells transfected with cDNA of glycosylated lysozyme. The retention was insensitive to NH4Cl. In the secreted protein, lysozyme contained predominantly complex oligosaccharides as demonstrated by a proteolytic fragmentation in vitro and resistance to endo-β-N-acetylglucosaminidase H. In contrast, when targeted to lysosomes, the lysozyme moiety of the fusion protein contained predominantly mannose-rich oligosaccharides. In baby hamster kidney cells, the trimming of the oligosaccharides in the lysozyme fragment was less vigorous, and a selective targeting of molecules bearing mannose-rich oligosaccharides to lysosomes was more apparent than in Chinese hamster ovary cells. In the presence of monensin, the formation of complex oligosaccharides in the fusion protein and its secretion were strongly inhibited, whereas the intracellular fragmentation was not. We suggest that the prosequence of myeloperoxidase participates in the intracellular routing of the precursor and that this routing operates on precursors bearing mannose-rich rather than terminally glycosylated oligosaccharides and diverts them from the secretory pathway at a site proximal to the monensin-sensitive compartment of the Golgi apparatus. J. Cell. Biochem. 71:158-168, 1998. © 1998 Wiley-Liss, Inc.
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  • 48
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    Journal of Cellular Biochemistry 71 (1998), S. 169-181 
    ISSN: 0730-2312
    Keywords: intermediate filaments ; mitogen-activated protein ; kinase-activated protein kinase-2 ; vimentin ; okadaic acid ; phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Organization of intermediate filament, a major component of cytoskeleton, is regulated by protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved protein kinases and phosphatases. Blocking dephosphorylation by protein phosphatase inhibitors such as okadaic acid (OA) leads to an apparent activation of protein kinase(s) and to genuine activation of phosphatase-regulated protein kinase(s). Treatment of 9L rat brain tumor cells with OA results in a drastically increased phosphorylation of vimentin, an intermediate filament protein. In-gel renaturing assays and in vitro kinase assays using vimentin as the exogenous substrate indicate that certain protein kinase(s) is activated in OA-treated cells. With specific protein kinase inhibitors, we show the possible involvement of the cdc2 kinase- and p38 mitogen-activated protein kinase (p38MAPK)-mediated pathways in this process. Subsequent in vitro assays demonstrate that vimentin may serve as an excellent substrate for MAPK-activated protein kinase-2 (MAPKAPK-2), the downstream effector of p38MAPK, and that MAPKAPK-2 is activated with OA treatment. Comparative analysis of tryptic phosphopeptide maps also indicates that corresponding phosphopeptides emerged in vimentin from OA-treated cells and were phosphorylated by MAPKAPK-2. Taken together, the results clearly demonstrate that MAPKAPK-2 may function as a vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38MAPK signaling cascade, via MAPKAPK-2, in the maintenance of integrity and possible physiological regulation of intermediate filaments. J. Cell. Biochem. 71:169-181, 1998. © 1998 Wiley-Liss, Inc.
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  • 49
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    Journal of Cellular Biochemistry 71 (1998), S. 149-157 
    ISSN: 0730-2312
    Keywords: RAP ; α2MR/LRP ; melanocytes ; melanoma ; cell culture density ; flow cytometry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: α2-Macroglobulin receptor/low-density lipoprotein receptor-related protein is a multifunctional cell surface receptor known to bind and internalize a large number of ligands. α2-Macroglobulin receptor-associated protein acts as an intracellular “chaperone” for this receptor, and it has been shown to inhibit binding of all its known ligands. In this paper, we characterize the expression of the receptor-associated protein in both normal human epidermal melanocytes and in six different human melanoma cell lines, by the use of flow cytometry and Western blotting analysis. We show that all the melanoma cell lines and the normal melanocytes express the receptor-associated protein at similar levels, with most located intracellularly. No receptor-associated protein was detected at the cell surface in the melanocytes or in three of the cell lines. However, in two of the melanoma cell lines, large amounts of receptor-associated protein were found on the cell surface, these having the largest amounts of it reported to date; in a further melanoma cell line, there was a small amount at the cell surface. We have also shown that the melanocytes and all the melanoma cell lines express the receptor itself at a wide range of levels, the highest levels of both the cell surface receptor and the cell surface receptor-associated protein being found in one particular melanoma cell line. By growing the cell lines under controlled conditions, we have demonstrated that, although the total cellular content of the receptor is markedly increased at high cell culture density, this treatment has no effect on the level of expression of the receptor-associated protein. J. Cell. Biochem. 71:149-157, 1998. © 1998 Wiley-Liss, Inc.
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  • 50
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    Journal of Cellular Biochemistry 71 (1998), S. 189-203 
    ISSN: 0730-2312
    Keywords: glucose transporters ; sperm ; dehydroascorbic acid ; fructose ; 2-deoxy-D-glucose ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We analyzed the expression of hexose transporters in human testis and in human, rat, and bull spermatozoa and studied the uptake of hexoses and vitamin C in bull spermatozoa. Immunocytochemical and reverse transcription-polymerase chain reaction analyses demonstrated that adult human testis expressed the hexose transporters GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5. Immunoblotting experiments demonstrated the presence of proteins of about 50-70 kD reactive with anti-GLUT1, GLUT2, GLUT3, and GLUT5 in membranes prepared from human spermatozoa, but no proteins reactive with GLUT4 antibodies were detected. Immunolocalization experiments confirmed the presence of GLUT1, GLUT2, GLUT3, GLUT5, and low levels of GLUT4 in human, rat, and bull spermatozoa. Each transporter isoform showed a typical subcellular localization in the head and the sperm tail. In the tail, GLUT3 and GLUT5 were present at the level of the middle piece in the three species examined, GLUT1 was present in the principal piece, and the localization of GLUT2 differed according of the species examined. Bull spermatozoa transported deoxyglucose, fructose, and the oxidized form of vitamin C, dehydroascorbic acid. Transport of deoxyglucose and dehydroascorbic acid was inhibited by cytochalasin B, indicating the direct participation of facilitative hexose transporters in the transport of both substrates by bull spermatozoa. Transport of fructose was not affected by cytochalasin B, which is consistent for an important role for GLUT5 in the transport of fructose in these cells. The data show that human, rat, and bull spermatozoa express several hexose transporter isoforms that allow for the efficient uptake of glucose, fructose, and dehydroascorbic acid by these cells. J. Cell. Biochem. 71:189-203, 1998. © 1998 Wiley-Liss, Inc.
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  • 51
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    Journal of Cellular Biochemistry 71 (1998), S. 182-188 
    ISSN: 0730-2312
    Keywords: human islets ; insulin release ; sulfonylurea receptors ; oral antidiabetic compounds ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic β cells or stable β-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characterisitics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [3H]glibenclamide saturable binding was shown to be linear at ≤0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25°C indicated the presence of a single class of saturable, high-affinity binding sites with a Kd value of 1.0 ± 0.07 nM and a Bmax value of 657 ± 48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride 〉 tolbutamide 〉 chlorpropamide ≫ metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds. J. Cell. Biochem. 71:182-188, 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. 204-215 
    ISSN: 0730-2312
    Keywords: osteoclast ; spectrin ; membrane skeleton ; bone ; bone resorption ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The presence of spectrin was demonstrated in chick osteoclasts by Western blotting and light and electron microscopic immunolocalization. Additionally, screening of a chick osteoclast cDNA library revealed the presence of α-spectrin. Light microscope level immunocytochemical staining of osteoclasts in situ revealed spectrin staining throughout the cytoplasm with heavier staining found at the marrow-facing cell margin and around the nuclei. Confocal microscopy of isolated osteoclasts plated onto a glass substrate showed that spectrin encircled the organelle-rich cell center. Nuclei and cytoplasmic inclusions were also stained and the plasma membrane was stained in a nonuniform, patchy distribution corresponding to regions of apparent membrane ruffling. Ultracytochemical localization showed spectrin to be found at the plasma membrane and distributed throughout the cytoplasm with especially intense staining of the nuclear membrane and filaments within the nuclear compartment. J. Cell. Biochem. 71:204-215, 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. 216-232 
    ISSN: 0730-2312
    Keywords: assembly of type I collagen ; COOH-terminal propeptide ; pepsin-resistant heterotrimers ; interspecies collagen molecule ; thermal stability ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Procollagen (Type I) contains a noncollagenous COOH-terminal propeptide (C-propeptide) hypothesized to be important in directing chain association and alignment during assembly. We previously expressed human pro-α2(I) cDNA in rat liver epithelial cells, W8, that produce only pro-α1(I) trimer collagen (Lim et al. [1994] MatrixBiol. 14: 21-30). In the resulting cell lines, α2(I) assembled with α1(I) forming heterotrimers. Using this cell system, we investigated the importance of the COOH-terminal propeptide sequence of the pro-α2(I) chain for normal assembly of type I collagen. Full-length human pro-α2(I) cDNA was cloned into expression vectors with a premature stop signal eliminating the final 10 amino acids. No triple-helical molecules containing α2(I) were detected in transfected W8 cells, although pro-α2(I) mRNA was detected. Additional protein analysis demonstrated that these cells synthesize small amounts of truncated pro-α2(I) chains detected by immunoprecipitation with a pro-α2(I) antibody. In addition, since the human-rat collagen was less thermostable than normal intraspecies collagen, wild-type and C-terminal truncated mouse cDNAs were expressed in mouse D2 cells, which produced only type I trimers. Results from both systems were consistent, suggesting that the last 10 amino acid residues of the pro-α2(I) chain are important for formation of stable type I collagen. J. Cell. Biochem. 71:216-232, 1998. © 1998 Wiley-Liss, Inc.
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  • 54
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 70 (1998), S. 222-230 
    ISSN: 0730-2312
    Keywords: functional organization of the nucleus ; nucleolus ; speckled compartment ; targeting sequence ; DNA replication ; RNA splicing ; nuclear matrix ; cell cycle ; DNA methyltransferase ; DNA ligase I ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Though there are no separating membranes within the nucleus, different factors are often concentrated at sites where their respective function is required, a phenomenum referred to as functional organization of the nucleus. How is then this organization achieved and how are the different metabolic processes integrated in the nucleus? One emerging principle was revealed by the identification of protein domains that, though not involved in catalysis, regulate enzyme activity at a higher order level by targeting enzymes to the right place at the right time. These targeting sequences constitute an assembly code for nuclear ‘protein factories,’ which ensure the extremely high efficiency and accuracy needed in a complex and competitive environment as the living mammalian cell. J. Cell. Biochem. 70:222- 230, 1998. © 1998 Wiley-Liss, Inc.
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  • 55
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    New York, N.Y. : Wiley-Blackwell
    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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