摘要:

AbstractStructural lesions in cAMP‐binding sites of regulartory (R) subunit of cAMP‐dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide‐depencient kinase activation in preparations from cells that were hemizygous or heterozygous for mutant RIsubunit expression. No wild‐type kinase activation was observed in extracts from heterozygous mutant cells. This “dominance” was investigated by characterizing expression of wild‐type and mutant RIsubunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild‐type RIsubunits and 3, subunits with a lesion (Glu200) tnat inactivate:. cAMP‐binding site A. By both studies of cAMP dissociation and two‐dimensional gel analysis, wild‐type R subunits comprised about 35% of total RIsubunits in heterozygous mutants. Synthesis of wild‐type and mutant RIsubunits was equivalent, but wild‐type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel RIsubunit‐containingspeciesfromheterozygousmutantpreparationswhoseelution behavior suggested a trimeric kinase consisting of an RIsubunit dimer and one catalytic (C) subunit. Wild‐type RIsubunit was found only in dimer and “trimer” peaks; the tetrameric kinase peak contained only mutant RIsubunit. It is concluded that C subunit binds preferentially to mutant RIsubunit in heterozygous cells forming either tetrameric kinase with mutant RIsubunit homodimers or trimeric kinase with RIsubunit heterodimers. This preferential binding results both suppression of wild‐type kinase activation and differential

ISSN:0021-9541    

DOI:10.1002/jcp.1041460112

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractExtracellular calcium (Cao) and the steroid hormone 1,25(OH)2D, induce the differentiation of human epidermal cells in culture. Recent studies suggest that increases in intracellular free calcium (Cai) levels may be an initial signal that triggers keratinocyte differentiation. In the present study, we evaluated cornified envelope formation, the terminal event during keratinocyte differentiation, and correlated it with changes in the Cai levels during differentiation of keratinocytes in culture induced by Cao or 1,25(OH)2D, Keratinocytes were grown in different Cao concentrations (0.1 or 1.2 mM) or in the presence of 1,25(OH)2D OCT'1 to 10−7M, and the Cai levels were measured using the fluorescent probe lndo‐1. Our results suggest that the induction of cornified envelope formation is associated with an increase in Cai level during calcium‐induced differentiation. Cao and the calcium ionophore ionomycin acutely increased Cai and cornified envelope formation. In contrast, the effect of 1,25(OH)2D on increasing Cai levels and stimulating cornified envelope formation was long‐term, requiring days of treatment with 1,25(OH)2D. Our data are consistent with other recent studies and support the hypothesis that Cao regulates keratinocyte differentiation primarily by acutely increasingtheirCai levels. The roleofcalcium in the mechanism ofaction of 1,25(OH)2D keratinocyte differentiation is less clear. The increase in Cai of keratinocytes during 1,25(OH)2D induced differentiation may be essential for or subsequent to its prodifferentiation

ISSN:0021-9541    

DOI:10.1002/jcp.1041460113

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractAD4743 is an antidiabetic agent that, when added to fetal bovine serum (FBS), has been shown to have adipogenic activity for some proadipocyte cell lines once they reach confluence. In the current study, the effects of AD4743 on the growth and adipocytic differentiation of 3T3 T multipotential mesenchymal stem cells have been tested. 3T3 T cells, unlike other cells capable of undergoing adipocyte differentiation, are routinely induced to differentiate at low cell density. This is done using platelet‐poor human plasma (HP), a potent inducer of growth arrest and differentiation. AD4743 (0–200 μg/ml) was tested in varied concentrations of HP or FBS, at varied cell densities, and at various times during growth and differentiation. AD4743 slowed the growth rate of 3T3 T cells and it induced their differentiation in a dose‐dependent manner in medium containing 10% FBS once they reached confluence. Tiie data suggest that the ability of AD4743 to inhibit growth may also becoupled with its ability to enhance differentiation. In addition, AD4743 (1–10 μg/ml) in the presence of 25% HP markedly increased the kinetics of adipocyte differentiation, at low (<5,000 cells/cm2) or high cell density. Greater than 50% cell differentiation could be achieved in 2 days in low density cultures; 80–95% differentiation could be achieved in just 4 days, compared to 8–12 days in a typical culture. The maximum amount of differentiation in HP was potentiated by AD4743 to agreater degree in poor lots of HP; however, the kinetics were increased in all lots. Adipocytic differentiation was measured both morphologically and by Northern blot analyses of differentiation‐specific genes. AD4743 at 1–10 μg/ml appeared to be most effective, depending on the cell density and other conditions. The mechanism of action of AD4743 remains to be elucidated, but the enhancement of adipocyte differentiation does not appear to occur via an insulin

ISSN:0021-9541    

DOI:10.1002/jcp.1041460114

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe contraction of collagen lattices made with arterial smooth muscle cells was studied in medium MCDB 107 without serum or supplemented with 1% fetal bovine serum, plus insulin, transferrin, and low‐density lipoprotein. Under these conditions, smooth muscle cell mitogens including HBGF‐1 (aFGF), PDGF, and EGF stimulated contraction. Stimulation by HBGF‐1 was more profound than with other factors tested. HBGF‐1 stimulation of lattice contraction was blocked by protein synthesis inhibitors, but not by inhibitors of DNA synthesis. Histological observations indicated that HBGF‐1 also enhanced the maintenance of healthy cells in the lattice. Taken together, these observations suggest that HBGF‐1 stimulates lattice contraction, not by a mitogenic effect, but by stimulating synthesis of specific cellular proteins. Since the greatest effects of HBGF‐1 on lattice contraction were seen during the first 72 h following casting, the effects on maintenance of cell viability are probably less important in promoting lattic

ISSN:0021-9541    

DOI:10.1002/jcp.1041460115

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractRecent studies indicate that the cytoskeleton may be involved in modulating tissue‐specific gene expression in mammalian cells. We have studied the role of the cytoskeleton in regulating milk protein synthesis and secretion by primary mouse mammary epithelial cells cultured on a reconstituted basement membrane that promotes differentiation. After 8 days in culture, cells were treated with cytochalasin D (CD) (0.5–1 μg/ml) to alter actin filaments or acrylamide (Ac) (5 mM) to alter intermediate filaments (cytokeratins). CD inhibited synthesis of most proteins in a concentration‐dependent manner, with β‐casein being the first affected. In contrast, Ac increased protein synthesis and secretion by 17–31% after a 12 hr treatment. Polyacrylamide gel electrophoresis of total secreted proteins indicates that synthetic rates of most proteins were increased equally by Ac treatment. This increase is apparently controlled at the level of translation, because control and Ac‐treated cells contained the same amount of poly‐A+RNA, and neither CD nor Ac altered mRNA levels for β‐casein. There was also no indication that either CD or Ac can induce the expression of milk proteins in quiescent cells cultured on a plastic substratum. In conjunction with the biochemical studies, changes in cytoskeletal morphology caused by the drug treatments were analyzed by immunofluorescence microscopy. As has been observed in other cell types, low concentrations of CD caused cells to round up by disrupting actin filaments. Ac treatment slightly decreased the intensity of actin staining, but no changes in microfilament organization were observed. Ac‐treated cells showed slight disorganization of the cytokeratin filaments, with some peripheral interfibrillar bundling, but the cytokeratin network did not collapse and no retraction of cell extensions or breakdown of cell‐cell contacts was observed. These results confirm previous reports that the actin cytoskeleton may play a role in regulating tissue‐specific protein synthesis. How Ac stimulates protein synthesis is unknown, but it is unlikely that this effect is directly mediated throug

ISSN:0021-9541    

DOI:10.1002/jcp.1041460116

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractHuman glomerular epithelial cells (GECs) in culture synthesize single‐chain, urokinase‐type plasminogen activator (SC‐uPA), tissue‐type plasminogen activator (t‐PA), and plasminogen activator inhibitor 1 (PAI‐1) and possess specific membrane‐binding sites for u‐PA. Using purified125I‐alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 ± 1.0 × 10−10M, 5.4 ± 1.4 × 104M sites per cell, 2. Kd = 1.6 ± 0.5 × 10−8M, 7.9 ± 1.8 × 105sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time‐and dose‐dependent increase of SC‐uPA, t‐PA, and PAI‐1 antigens released by GECs. Thrombin‐mediated increase in antigen was paralleled by an increase in the levels of corresponding u‐PA and PAI‐1 messenger RNA. In contrast, thrombin decreased u‐PA activity in conditioned medium. This discrepancy between u‐PA antigen and u‐PA activity was explained by a limited proteolysis of SC‐uPA by thrombin, leading to a two‐chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u‐PA binding sites on GECs (p<0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persis

ISSN:0021-9541    

DOI:10.1002/jcp.1041460117

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractProstaglandin E2(PGE2, 5 ng/ml to 5 μg/ml) induced a dose‐dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+([Ca2+]i) in a clonal osteoblast‐like cell line, MOB 3–4. In contrast, prostaglandin F2α (PGF2α, 5 ng/ml to 5 μg/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2(>0.5 μg/ml) and PGF2α (≥0.5 μg/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF‐loaded cells. A tumor promotor, phorbol 12‐myristate 13‐acetate (PMA, 0.1–100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2‐(5 μg/ml) and PMA‐ (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+exchange, or H‐7 (100 μM), a nonspecific inhibitor of protein kinase C. Thus, MOB 3–4 cells appeared to possess PGE2receptors and PGF2α receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride‐sensitive Na+/H+exchange activity, which increases pHi in response to PGE2and PGF2α, as well as to PMA. Long‐term (48 hr) exposure of the cells to PGE2at a high concentration (5 μg/ml), but not to PGF2α and PMA, decreased DNA synthesis in the serum‐deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3–4 cells, the inhibitory effect of PGE2on DNA synth

ISSN:0021-9541    

DOI:10.1002/jcp.1041460118

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe effect of calf blood extract (Solcoseryl, SS) on mitochondrial oxidative function in various states was studied polarographically in vitro. (1) Mitochondrial respiration in all 4 conventional study states (Estabrook, 1967) was enhanced by the addition of SS, including states 1 and 2 (endogenous substrates only). (2) The effect of SS on mitochondrial oxygen consumption was concentration dependent, while ADP/O ratio remained constant. The effect of added respiratory substrates varied with the particular substrate at optimally active concentrations. With suboptimal substrate levels, ADP/O ratios were concentration dependent, in contrast to the SS effect. Under oligomycin ATPase inhibition, SS was no longer active, in contrast to DNP, which remained active. (3) In states 3 (added ADP) and 4 (ADP exhausted), oxygen consumption and oxidative phosphorylation were enhanced by SS in the presence or absence of citrate, glutamate, pyruvate, lactate, or ascorbate. However, in the presence of succinate, SS had no effect. (4) ADP/O ratio was decreased by SS in the presence of added substrate, suggesting that SS activation of H+‐ATPase enhances ATP hydrolysis as well as oxidative phosphorylation and ATP synthesis. (5) The enhancing effect of SS on mitochondrial function is due to hydrophilic components of SS. The lipidic components obtained by Folch fraction of SS have no effect. It is concluded that the effects of SS respiratory substrates and uncouplers on mitochondrial function are essentially different. SS enhances both ATP synthesis and oxygen consumption by mitochondri

ISSN:0021-9541    

DOI:10.1002/jcp.1041460119

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractHuman cellular polypeptide factors, namely interferon‐α, interferon‐γ, transforming growth factor (TGF)‐α, and TGF‐β, were analyzed for their effect on motility of human sperm cells. Both interferons caused an inhibition of sperm cell motility due to direct cytotoxic effects without inducing 2′‐5′ oligoadenylate [2–5(A)]synthetase activity. TGF‐α affected neither motility nor the levels of 2–5(A) synthetase in sperm cells. TGF‐β, had no affect on sperm motility, yet it caused an induction of 2–5(A)synthetase activity. Western immunoblot analysis of TGF‐β1‐treated sperm indicated an enhancement of a 50 kDa protein. Metabolic labeling of sperm cells revealed biosynthesis of one major protein of 50 kDa and at least five minor proteins in the range of 30–92 kDa; the level of 50 kDa protein increased after treatment with TGF‐β1. The treatment of sperm cells with TGF‐β1did not affect their penetration in zona‐free hamster eggs (SPA). These results indicate that TGF‐β1enhances expression of a 50 kDa protein related to 2–5(A)synthetase in human sperm cells along with other minor proteins, and thi

ISSN:0021-9541    

DOI:10.1002/jcp.1041460120

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe formation of blood vessels in vivo (angiogenesis) is an important process and is usually initiated in response to injury, tumor growth, or normal tissue development. We have studied the effect of human interferon (IFN) alpha (α) and gamma (γ) on the capillary‐like network formation in an in vitro model of angiogenesis using human umbilical vein endothelial cells (HUVEC). When HUVEC cells are plated on Matrigel (reconstituted basement membrane matrix enriched in laminin), a network of capillary like structures (endotubes) rapidly forms. IFN‐α enhanced the tube formation in a dose‐dependent manner, whereas IFN‐γ significantly inhibited the tube formation. In addition, both the enhancement and inhibition of angiogenesis by IFN‐α and γ was found to be greater if the cells were pretreated with IFN for 12 hr before plating on the Matrigel. These results suggest that IFN may play an important role in several vascular processes including early stages of wound healing, recanalization of thrombi, tumor growth, metastasis, normal growth,

ISSN:0021-9541    

DOI:10.1002/jcp.1041460121

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY