摘要:

AbstractWhen nuclei were isolated from Chinese hamster ovary cells after being heated, there was a large increase in the amount of3H‐tryptophan labeled nonhistone protein in the nucleusrelativeto the whole cell. After 15 min or 30 min of heating at 45.5°C, the nuclear nonhistone protein content increased by 1.6 or 1.8, respectively. In contrast, when the nuclear nonhistone protein content was determined in theintactcell by using autoradiography to quantify3H‐tryptophan labeled protein in the nucleus and cytoplasm in sections offixedcells, the nuclear nonhistone protein content increased by only 1.14 or 1.28 for 15 or 30 min at 45.5°C, respectively. Therefore, heat does not induce a massive movement of cytoplasmic protein into the nucleus. © 1993 Wiley‐L

ISSN:0021-9541    

DOI:10.1002/jcp.1041540202

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe intracellular pathway following receptor‐mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)‐elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID50value similar to the LD50of BFA in Y1 adrenal cells. Binding and internalization of [125I]‐cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA‐sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK1cells, of which the Golgi structure was BFA‐resistant. These results strongly suggest that a BFA‐sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin,Pseudomonastoxin, and modeccin, even though their structures and modes of action are very different. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041540203

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

年代:1993

数据来源: WILEY

摘要:

AbstractChronic anoxia, glucose starvation, low pH, and numerous other conditions induce the glucose‐regulated system of stress proteins (GRPs), whose principal members are observed at 78, 94, and 170 kDa. These stresses may be expected to occur during growth in untreated tumors. To examine the possibility that GRPs are correspondingly induced, we have examined the protein profiles of small (1.8 g) radiation‐induced fibrosarcoma (RIF) tumors grown on C3H mice. One and two‐dimensional gel electrophoresis indicate that the principal GRPs at 78 and 94 are coordinately and substantially increased in large tumor masses, relative to the small, and may be partially increased in the intermediate tumors. Necrotic material removed from large tumors exhibited an identical pattern of GRP induction with no visible indication of protein degradation and also contained a significant fraction of viable cells. Western blot analysis using rabbit antisera raised against the 78 and 170 kDa GRPs also demonstrated the enhanced accumulation of these proteins in the large tumors. The antibody against the 170 kDa GRP was also capable of detecting the induction of this stress protein in large tumors by indirect immunofluorescence analysis. Northern blot studies using a probe for the GRP 78 gene also showed an increase in GRP 78 message in large tumors as well as in RIF cells exposed to anoxic stress in vitro. Two‐dimensional gel electrophoresis indicated that the major heat shock proteins at 70 and 90 kDa were not increased in the larger tumors, and the amount of the 90 kDa species was reduced. Finally, the quantity of vimentin and its degradation products is significantly diminished in large tumors and in anoxic cells. This study demonstrates that RIF tumor cells undergo a glucose regulated stress response in situ during tumor growth. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041540204

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

年代:1993

数据来源: WILEY

摘要:

AbstractAddition of polyamines or their analogs to newly confluent LLC‐PK1cells resulted in down‐regulation of Na+‐glucose transport (symport) activity. Polyamines prevented the induction of this symporter by the differentiation inducer hexamethylene bisacetamide (HMBA) but did not influence induction by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX). Partial depletion of endogenous polyamines after addition of α ‐difluoromethylornithine (DFMO) resulted in a 4 to 5‐fold increase in symporter expression. Symporter induction by either HMBA or DFMO was inhibited by the protein kinase inhibitor H‐7 but H‐7 did not affect symporter induction by IBMX. Changes in symporter activity were accompanied by changes in levels of the 75 kD symporter subunit detected by Western blot. Cultures exposed to HMBA exhibited reduced levels of ornithine decarboxylase activity. Our results suggest that induction of symporter expression by HMBA may be mediated in part by its effects on polymine metabolism, and point to parallel roles of polyamines and cyclic AMP in regulating the expression of this physiologically important renal transport system. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041540205

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

年代:1993

数据来源: WILEY

摘要:

AbstractIsolated rat hepatocytes were exposed to hypotonic media (225 mosmol/l) for 5 and 15 min and processed for a quantitative electron microscopic stereologic analysis. Within 5 min of hypotonicity, the hepatocyte volume increased by 25% and thereatter displayed a volume regulatory decrease leading to mean cellular volume, which was 16% above that of controls. Stereologic analysis of the major subcellular compartment, the cytosol, showed an identical change as the whole cell. In contrast to that, the mitochondrial compartment increased in volume by 30% within the first 5 min of exposure and returned by regulatory volume decrease back to values of the isotonic controls after 15 min of hypotonicity. In contrast, hypotonicity (220 mosmol/l)‐stimulation of flux through mitochondrial glutaminase and the glycine cleavage enzyme complex, as assessed by14CO2production from [1‐14C]glutamine or [1‐14C]glycine in isolated perfused rat liver persisted throughout a 15‐min period of hypotonic exposure. Thus hypotonicity‐induced alterations of mitochondrial metabolism apparently do not parallel the time course of mitochondrial volume changes. This suggests that persistent mitochondrial swelling is not required for functional alterations, but that the latter may be triggered by the initial swelling of mitochondria. Hypotonic exposure did not alter the nuclear volume of isolated hepatocytes. Cell membrane surface nearly doubled after 5 min of hypotonic exposure, but returned within 15 min of exposure to values observed in normotonic media. This may reflect the participation of exocytosis in hepatocyte volume regulation. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041540206

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe androgen‐dependent clonal cell line SC‐3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)‐autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC‐3 cells, FGF receptor expression is upregulated by the SC‐3‐derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effect of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC‐3 cells were studied. Suramin inhibited androgen‐dependent growth of SC‐3 cells in a concentration‐dependent fashion: ∼50% inhibition was observed at 25 μM. [3H]Thymidine incorporation into the cells stimulated with partially purified SC‐3‐derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF‐induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC‐3 cells with suramin decreased cell surface125I‐bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 μM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor‐1 mRNA to a similar extent, whereas it appeared not to affect the levels of β‐actin mRNA. Moreover, suramin completely blocked androgen‐ or bFGF‐induced accumulation of FGF receptor‐1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC‐3 cells through inhibition of an androgen‐inducible autocrine loop involving SC‐3‐derived gr

ISSN:0021-9541    

DOI:10.1002/jcp.1041540207

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

年代:1993

数据来源: WILEY

摘要:

AbstractChondrocytes in cartilage are embedded in a matrix containing a high concentration of proteoglycans and hence of fixed negative charges. Their extracellular ionic environment is thus different from that of most cells, with extracellular Na+being 250–350 mM and extracellular osmolality 350–450 mOsm. When chondrocytes are isolated from the matrix and incubated in standard culture medium (DMEM; osmolality 250–280 mOsm), their extracellular environment changes sharply. We incubated isolated bovine articular chondrocytes and cartilage slices in DMEM whose osmolity was altered over the range 250–450 mOsm by Na+or sucrose addition.35S‐sulphate and3H‐proline incorporation rates were at a maximum when the extracellular osmolality was 350–400 mOsm for both freshly isolated chondrocytes and for chondrocytes in cartilage. The incorporation rate per cell of isolated chondrocytes was only 10% that of chondrocytes in situ both 4 and 24 hours after isolation. For freshly isolated chondrocytes, the rate increased 30–50% in DMEM to which NaCl or sucrose had been added to the increase osmolality. In chondrocytes incubated overnight in DMEM, the rate was greatest in DMEM of normal osmolality and fell from the maximum in proportion to the change in osmolality. The effects of surcrose addition on incorporation rates were similar but not identical to those of Na+addition. Changes in cell volume might be linked to changes in synthesis rates since the cell volume of chondrocytes (measured by Coulter‐counter) increased 30–40% when the cells are removed from their in situ environment into DMEM. Synthesis rates can thus be partly regulated by changes in extracellular osmolality, which in cartilage is controlled by proteoglycan concentration. This provides a mechanism by which the chondrocytes can rapidly respond to changes in extracellular matrix composition. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041540208

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe studied the effect of cell age on the cation transport systems of rabbit erythrocytes by increasing the proportion of circulating young erythrocytes with either repeated bleeding or with phenylhydrazine (PHZ) treatment. We found that when the reticulocyte content of rabbit blood is increased by bleeding (from 1 to 40–50% of the circulating red cells), the response of the various transport pathways differs. The largest increase (fivefold) was found in the activity of K‐CI contransport which peaked 3 days after the last bleeding. The Na‐K pump activity peaked at a similar time, but the % increase was twofold less than the K‐CI contransport. There was very small increase in the activity of the Na‐Li exchange, whereas the Na‐H exchange reached peak values 10 days after the last bleeding (twofold increase), when activities of K‐Cl contransport and Na‐K pump had returned to almost normal levels. In vivo PHZ treatment resulted in anemia and marked reticulocytosis (80–90% of circulating cells). Transport rates were markedly increased (Na‐K pump 9.6‐fold, Na‐H exchange 6.8‐fold, Na‐Li exchange 2.75‐fold; K‐CI contransport: 10–20‐fold). When blood from PHZ‐treated rabbits was incubated in vitro for 24–48 hour, red cell volume and K content decreased. This process was associated with a 70% reduction in the activity of the K‐CI contransport after 24 hours and a 90% reduction after 48 hours. The activity of the other systems also declined and approached baseline values after 48 hours. Loss of transport activity was not affected by 10 μM E‐64, whereas 10 mM methylamine reduced the inactivation of the Na‐H exchange and of the Na‐Li exchange. PHZ treatment of rabbit red cells in vitro resulted in marked increase of the K‐CI contransport and inhibition of Na‐K pump, Na‐H exchange, and Na‐Li exchange. These effects were abolished by DTT, with the exception of the Na‐K pump inhibition, which was DTT insensitive. Thus both cell age and oxidative damage are important determinants of ca

ISSN:0021-9541    

DOI:10.1002/jcp.1041540209

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

年代:1993

数据来源: WILEY

摘要:

AbstractProgressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. We recently found that cultured smooth muscle cells (SMC) derived from arteries of patients with moyamoya disease responded poorly to serum mitogens, especially to platelet‐derived growth factor (PDGF). In the present study, we investigated further the binding and processing of125I‐PDGF, as well as down‐regulation of the PDGF receptors in arterial SMC derived from patients with moyamoya disease. The specific binding sites of125I‐PDGF were reduced significantly at both 4°C and 22°C on SMC from moyamoya disease compared with those from controls (4.78 vs. 11.92 × 104/cell at 4°C), though the apparen dissociation constant (Kd) were the same. Kinetics of125I‐PDGF binding at 37°C in cells from moyamoya disease showed fewer binding sites (less than 1/3 of controls) and lower degradation per cell than in those from controls, though no difference was observed in either internalization or degradation of each receptor. When SMC were exposed to lower concentrations of nonlabeled PDGF at 37°C, the percentage of remaining binding sites on cells from moyamoya disease was significantly less than that from controls. This excess down‐regulation of PDGF receptor in SMC from moyamoya disease may be interpreted as insufficent recycling or a decreased intracellular pool of the PDGF receptor. These results are closely correlated with the diminished proliferation responses to PDGF in SMC from moyamoya disease and provide evidence that functional alterations in vascular cells are involved in the mechanism of development of intimal thickening in moyamoya disease. © 19

ISSN:0021-9541    

DOI:10.1002/jcp.1041540210

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

年代:1993

数据来源: WILEY

摘要:

AbstractMacrophages may modulate mesangial expansion following renal injury via secretory products. We undertook the present study to determine the effects of macrophages supernatants on mesangial cell proliferation. Macrophage supernatants collected in serum‐free media after 24 hours caused significantly enhanced mesangial cell proliferation in long‐term culture at concentration up to 50% but caused suppression at higher concentration (control, 122,000 ± 14,000 cells/well: 50% supernatant, 188,000 ± 15,100 cells/well,p<0.02 compared to control, n = 4; 80% supernatant, 52,000 ± 3,500 cells/well,P<0.01 compared to control, n = 4). In short‐term culture [3H] thymidine incorporation, a measure of DNA synthesis, was significantly enhanced compared to control at supernatant concentrations up to 30% (30% supernatant, 4,120 ± 310 cpm/well; control, 3,210 ± 97 cpm/well,P<0.5, n = 4), but uptake was reduced at high concentration (80% supernatant, 2,900 ± 74 cpm/well; control, 3,210 ± 97 cpm/well,P<0.05, n= 4) When macrophages supernatants were collected after 48 hours incubation and incubated with mesangial cells, mesangial cell thymidine uptake was significantly suppressed compared to control (48‐hours supernatant, 4,060 ± 260 cpm/well; control, 5,890 ± 270 cpm/well,P<0.01, n = 4) and comapared to 24‐hour supernatants, which enhanced uptake (24‐hour supernatant, 8,080 ± 340 cpm/well; control, 5,890 ± 270 cpm/well,P<0.01, n =4). Our results suggest that macrophages supernatants can directly enhance mesangial cell proliferation in vitro in both short‐term and long‐term culture, though this effect is lost at high concentrations of supernatant. These data lend support to the potential role of the macrophage in mediating mesangial expansion following renal inju

ISSN:0021-9541    

DOI:10.1002/jcp.1041540211

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

年代:1993

数据来源: WILEY