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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 138 (1989), S. 367-374 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Black people have a higher propensity than caucasions toward essential hypertension. To explore the possibility that this racial difference relates to cellular Ca2+ metabolism, we measured 45Ca2+ washout and uptake and cytosolic free concentration of Ca2+ [Ca2+], in serially passed skin fibroblasts from normotensive black and white males. Depending on the experimental conditions, 45Ca2+ washout in these cells was described by either two or three exponential functions, whereas 45Ca2+ uptake was described only by a two-exponent function. There were no racial differences in 45Ca2+ uptake and washout of unstimulated fibroblasts. However, stimulation by human serum resulted in an increase in the 45Ca2+ washout that was higher in fibroblasts from blacks than from whites. The racial differences were expressed primarily by higher values of the apparent washout rate constant (k1) of 45Ca2+ from the largest and most rapidly exchangeable cellular pool. The effect of human serum was not related to its origin (blacks vs. whites). In 2 mM Ca2+ medium and 10% serum from blacks, the respective k1 (mean ± SEM; × 10-2/min) values for fibroblasts from blacks and whites were 89.68 ± 5.23 and 73.29 ± 4.0; in the presence of 10% serum from whites, the k1 values for cells from blacks and whites were 84.14 ± 2.80 and 76.36 ± 3.23 (overall significance of P .01). In Ca2+-deficient medium in the presence of 10% human serum, the k1 for fibroblasts from blacks and whites were 115.57 ± 3.76 and 102.15 ± 3.30 (P 〈 .05). Serum substantially increased the 45Ca2+ uptake in fibroblasts from both blacks and whites; however, racial differences were not observed. Basal levels of [Ca2+], were not different in fibroblasts of blacks vs. whites (46.8 ± 6.8 and 43.2 ± 7.1 nM for blacks and whites, respectively). However, the peak response of Ca2+ transients for cell stimulated by 5% human serum was significantly higher in blacks than whites (blacks = 963 ± 213, whites = 481 ± 162 nM; P = .0286). We conclude that Ca2+ regulation is different in serum-stimulated fibroblasts from blacks and whites and that, at least in part, this difference may relate to a greater agonist-induced mobilization of Ca2+ in fibroblasts from blacks.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 31 (1995), S. 273-282 
    ISSN: 0886-1544
    Keywords: Peritoneal macrophages ; F-actin microfilament ; in situ hybridization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In mouse fibroblasts, at least five TM isoforms are identified and they can be grouped into the high (TM1, TM2, and TM3) and low molecular weight TM isoforms (TM4 and TM5). Suppression of one of the high molecular weight tropomyosin (TM) isoforms in nonmuscle cells is implicated to be one of the causes for disorganization of actin microfilament bundles and subsequent changes in cell motility and cell shape. In this study, we studied the expression of tropomyosin isoforms in macrophages that exhibit high motility and ability to change cell shape. Two-dimensional gel electrophoresis followed by Western blot analysis using polyclonal anti-TM antiserum revealed that the high molecular weight TM isoforms were lacking in both resident and activated mouse peritoneal macrophages. Analyses of newly synthesized TM isoforms, Northern blot analyses using isoform-specific cDNA probes, and immunostaining with monoclonal anti-TM antibody that recognizes only the high molecular weight TM isoforms also demonstrated that the syntheses of the high molecular weight TM isoforms (TM1, TM2, and TM3) were completely suppressed, whereas the low molecular weight TM isoforms (TM4 and TM5) were expressed in macrophages. These results indicate that macrophages intrinsically lack the high molecular weight TM isoforms. In order to obtain information about cellular localization of the low molecular weight TM isoforms in macrophages, they were immunostained with polyclonal anti-TM antiserum that recognizes both the high and low molecular weight TM isoforms. The results showed that the low molecular weight TM isoforms were co-localized with F-actin in punctate and short fibrous structures. In addition, we performed in situ hybridization analysis to examine localizations of the TM mRNAs in fibroblasts and macrophages. The results showed that TM mRNAs were localized throughout the cytoplasm. © 1995 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 20 (1991), S. 38-46 
    ISSN: 0886-1544
    Keywords: cilia ; calcium ; cAMP ; differential response ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ciliated sheets of cell cortex were prepared from Triton-glycerol-extracted Paramecium to observe directly the change of ciliary orientation. The observation of the ciliary responses revealed the modes of ciliary control by Ca2+ and cyclic nucleotides. The cilia changed their pointing direction clockwise from 11-12 to 5 o'clock (with the anterior of the cell defined as 12 o'clock) in the horizontal plane of cell surface when Ca2+ concentration was decreased from 10-6 M to 10-7 M. Cyclic AMP competed with Ca2+ ion in determining the orientation of the cilia. On the other hand, cGMP tended to change the ciliary orientation toward 3 o'clock. Ciliary sensitivity to cyclic nucleotides depended on their location on the cell surface. The cilia on the left-hand field of the cell were more sensitive to cyclic nucleotide than those on the right-hand field. The differential distribution of ciliary sensitivity within a single cell seems to be functional in the sophisticated turning mechanism in the behavioral response of Paramecium.
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  • 4
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Prostaglandin (PG) F2α increased [3H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with ED50 values of 2.0 × 10-8 M, 4.6 × 10-8 M, and 7.5 × 10-8 M, respectively. The increase in [3H]thymidine incorporation with PGF2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca2+]i increase with PGF2α was still obvious in the absence of extracellular Ca2+ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF2α, which was sensitive to GTPγS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF2α-specific Cl- current when examined by voltage clamp. This Cl- current was also insensitive to IAP pretreatment and not affected by extracellular Ca2+ concentration ([Ca2+]o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF2α receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca2+ via an IAP-insensitive G-proteins(s), 2) that this PGF2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF2α receptor can be expressed in the oocyte system. © 1993 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 157 (1993), S. 461-468 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor-α (TGF-α). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin-8 and -18. But these cells expressed neither cytokeratin-7 nor-19. When 6 × 105 cells were plated on 35-mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF-α. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF + HGF, EGF + TGF-α, and HGF + TGF-α were not different from those of the colonies induced by each mitogen alone. To examine the ability of co-mitogenic factors to induce small-cell colonies, angiotensin-II, insulin-like growth factor-I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co-mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co-mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small-cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF-α, or FGFs and that the co-mitogens did not influence the formation of the small-cell colonies. © 1993 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 153 (1992), S. 1-5 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We investigated the effect of interleukin 6 (IL-6) on the migration of rabbit corneal epithelial in vitro and on the attachment of dissociated corneal epithelial cells to a fibronectin matrix. When corneal blocks were cultured with IL-6 for 24 hours, the length of the path of epithelial migration over exposed corneal stroma increased significantly (p 〈 0.005 at the concentration of 10 ng/ml) in proportion to the concentrations of IL-6 (0.1-10.0 ng/ml). The addition of antiserum against fibronectin or of GRGDSP abolished the stimulatory effect of IL-6 on epithelial migration. When corneal epithelial cells were cultured with various concentrations of IL-6, suspended, and plated on wells coated with fibronectin (10 μg/ml), the number of cells attached to the wells increased in a dose-dependent manner. The presence of antibody against fibronectin or of GRGDSP during the attachment assay decreased the number of cells attached to the fibronectin matrix, regardless of the fact that the cells had been cultured with IL-6 or not. IL-6 stimulated the attachment of corneal epithelial cells to collagen type IV and to laminin matrices. However, the presence of GRGDSP did not affect the cell attachment to collagen type IV and to laminin. These findings strongly indicate that IL-6 stimulates epithelial migration in the cornea by a fibronectin-dependent mechanism, presumably the increased expression of fibronectin receptors. © 1992 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 159 (1994), S. 213-220 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Endothelin-1 (ET-1) is an endothelium-derived 21 amino acid vasoconstrictor peptide possessing two intrachain disulfide bridges. Recently it has become evident that isoforms of ET (ET-1, -2, and -3) have a wide range of pharmacological effects in various tissues and act as autocrine/paracrine factors. We demonstrate here that ET-1 is secreted from normal human keratinocytes and may work as an autocrine growth factor through a specific receptor. In this study, human foreskin keratinocytes were cultured in serum-free MCDB 153 medium. Cell growth and [3H] thymidine incorporation in low and high Ca++ concentration media was stimulated by ET-1, -2, and -3 with similar potencies. The strongest response was observed at 10 nM ETs, whereas stimulatory activity was reduced at 100 nM. ETs suppressed keratinocyte differentiation as measured by reactivity with involucrin antibody. Plasminogen activator activity (mainly urokinase) in the medium was also stimulated by the addition of 10 nM ETs. ET-1-like immunoreactivity measured by radioimmunoassay was 1.4 fmol/day/106 cells in non-treated condition medium. Among the various cytokines, tumor necrosis factor-α (TNF-α), interleukin-1α, and transforming growth factor-β stimulated ET-1 secretion in a dose-dependent manner. The strongest response (ten-fold) was observed upon the addition of 10 ng/ml TNF-α. Scatchard plot analysis of [125I] ET-1 binding to keratinocytes revealed the presence of a single class of high affinity receptors (KD 50 pM, 9 x 103 sites/cell). Binding was competitively inhibited by the addition of unlabeled ET-1 and -2 with similar affinities and by ET-3 with weaker affinity. ET-1 mRNA expression in keratinocytes was detected by reverse transcription-polymerase chain reaction and was increased by treatment with 10 ng/ml TNF-α. These results suggest that ET-1 acts as an autocrine growth factor for keratinocytes through a specific receptor. © 1994 wiley-Liss, Inc.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 159 (1994), S. 415-422 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We wished to determine whether hyaluronan would affect the attachment of epithelial cells to extracellular matrix proteins. Multiwell tissue culture plates were coated with human plasma fibronectin, laminin, or collagen type IV (0.01-10.0 μg/ml). Single-cell suspensions of rabbit corneal epithelial cells were placed in the wells, and after 45 minutes incubation the cells adhering to the matrix proteins were stained and counted. Cells attached to all three types of proteins. Preincubation of the matrix proteins with hyaluronan (0.1-1.0 mg/ml) significantly increased the number of cells attached to the fibronectin matrix, but it did not increase the numbers of cells attached to laminin or collagen type IV. Hyaluronidase inhibited this stimulatory effect. Glycosaminoglcyans other than hyaluronan (chondroitin sulfate, keratan sulfate, or heparan sulfate) failed to increase the numbers of attached cells. Treatment of the fibronectin matrix with monoclonal antibodies against the cell-binding domain of fibronectin (FN12-8 or FN30-8, 0.03-0.3 mg/ml, for 1 hour), before or after hyaluronan treatment, significantly decreased the numbers of attached cells. Monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal (FN9-1), however, significantly decreased the number of attached cells only when this antibody treatment preceded the hyaluronan treatment. Preincubation of the cells with hyaluronan had no effect; preincubation with GRGDSP (1 mg/ml), a synthetic peptide that blocks the cell surface receptor for fibronectin, significantly decreased cell attachment whether the fibronectin matrix was treated with hyaluronan or not. Further studies demonstrated that monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin prevented radiolabeled hyaluronan from binding to fibronectin; likewise, the isolated N-terminal fragment, coupled with Sepharose 4B, bound to hyaluronan in columns. We conclude that hyaluronan binds to a fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin and facilitates the attachment of epithelial cells. © 1994 wiley-Liss, Inc.
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  • 9
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Osteoclast formation from hemopoietic precursors has been shown to require the support of stromal cells in bone tissue. In this study, we demonstrated that hepatocyte growth factor (HGF) is one of the stromal cell-derived molecules responsible for osteoclast-like cell formation. For our experimens, we used a coculture system for osteoclastic cell formation and activation in which hemopoietic blast cells are cocultured with calvaria-derived stromal MC3T3-G2/PA6 (PA6) cells on dentine slices in the presence of 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3]. Addition of anti-HGF neutralizing IgG to the cocultures inhibited the formation of osteoclastic cells and their dentine-recorbing activity. We detected a single 6.0-kb transcript for HGF in PA6 cells, and also recognized immunoreactive Mr. 81,000 and 88,000 forms of HGF in conditioned medium (CM) from PA6 cell cultures, the level of which reached 6 ng/ml. Both the CM and HGF stimulated the proliferation of blast cells synergistically with granulocyte-macrophage colony-stimulating factor, resulting in an increased number of osteoclast precursors that respond to 1,25(OH)2D3 that are tartrate-resistant acid phosphatase-positive multinucleate cells in stromal cell-free blast cell cultures in plastic wells. The effect of the CM was diminished by the addition of anti-HGF IgG. However, neither the CM nor HGF stimulated the formation of osteoclastic cells and pits on dentine slices in the absence of PA6 cells. These results suggest that although HGF cannot completely replace stromal cells, it is one of the paracrine mediators produced by stromal cells that act on proliferation of osteoclastic cell precursors. © 1995 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 167 (1996), S. 422-433 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are known to stimulate the locomotion of epithelial cells in culture. However, the molecular mechanisms which mediate these important changes are poorly understood. Here we have determined the effects of HGF and EGF on hepatocyte morphology, cytoskeletal organization, and the expression of molecular motor-encoding genes. Primary cultures of hepatocytes were treated with 10 ng/ml of HGF or EGF and observed with phase and fluorescence microscopy at 10, 24, and 48 h after treatment. We found that, over time, treated cells spread and became elongated after 24 h of treatment while forming long processes with dramatic alterations in the microtubule and actin cytoskeletons by 48 h. Quantitative Northern blot analysis was performed to measure expression of cytoskeletal-(β-actin, α-tubulin) and molecular motor-(dynein, kinesin, and myosin Iα and II) encoding genes which may contribute to this change in form. We observed the highest increase in levels of expression for myosin II (3.3-fold), kinesin (2.7-fold), myosin Iα (2.2- fold), and α-tubulin (1.9-fold) after only 2 h of treatment with HGF. In contrast, EGF upregulated the expression of myosin Iα (2.4-fold), kinesin (1.5-fold), and dynein (1.5-fold) at 10 h. The expression of the β-actin gene remained constant in HGF-treated cells, while EGF induced a slight upregulation after 10 h of treatment. These results show for the first time that a selective upregulation of molecular motor-encoding genes correlates with alterations in cell shape and motility induced by HGF and EGF. © 1996 Wiley-Liss, Inc.
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