bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitors of protein synthesis cause increased hexose transport in cultured human fibroblasts by a mechanism other than transporter translocation
Authors: Germinario Ralph J. , Manuel Susannia , Chang Zully , Leckett Blaine
Published: February 2005

We have investigated the effect of various inhibitors of protein synthesis on hexose transport in human skin fibroblasts using 2‐deoxy‐D‐glucose (2‐DG) and 3‐0‐methyl‐D‐glucose (3‐OMG) to measure hexose transport. Exposure of glucose‐fed, serum‐free cultures to cycloheximide (CHX) (50 μg/ml) for 6 h resulted in increased 2‐DG transport (3.81 ± .53 vs. 6.62 ± .88 nmoles/mg protein/2 min; n = 9) and 3‐OMG transport (1.36 ±.66 vs. 3.18 ± .83 nmoles/mg protein/30 sec; n = 4) in the CHX exposed group. Under these conditions inhibition of protein synthesis was greater than 90%. This CHX induced transport increase was time dependent (approaching maximum within 1 h of exposure to CHX) and related to an increase in the Vmax of hexose transport in the CHX exposed group (18.4 ± 2.4 vs. 4.8 ± 1.1 nmoles 2‐DG/mg protein/min) with no difference in the transport Km (1.55 ± .63 vs. 2.92 ± .59 mM). Further, the CHX induced increase in hexose transport was reversible. Exposure of human fibroblasts to inhibitors of protein synthesis with different mechanisms of action (e.g., puromycin, pactamy‐cin, or CHX) all generated hexose transport increases in a concentration‐dependent fashion correlating with their increasing inhibitory effects on protein synthesis. Nucleotidase enriched (i.e., plasma membrane) fractions of control and CHX‐exposed cells showed no differences in D‐glucose inhibitable cytochalasin B binding activity. Further, quantitative Western analysis of nucleotidase enriched fractions indicated CHX exposure resulted in no significant increase in glucose transporter mass compared with control plasma membrane fractions. Glucose deprived cells, however, which exhibited increased sugar transport comparable to the CHX‐exposed group, did show increased glucose transporter mass in the plasma membrane fraction. The data indicate that inhibitors of protein synthesis can cause a significant elevation in hexose transport and that the hexose transporter mass in the isolated plasma membrane fractions did not reflect the whole cell transport change. It is suggested that a mechanism other than glucose transporter translocation to the plasma membrane may be involved in causing this sugar transport increase. © 1992 Wiley‐Liss, Inc.
Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury
Authors: Bhat Ganesh B. , Tinsley Steven B. , Tolson J. Keith , Patel Jawaharlal M. , Block Edward R.
Published: February 2005

The effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2 challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time‐dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate‐treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia‐enhanced susceptibility to H2O2 injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2 injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2 challenge. © 1992 Wiley‐Liss, Inc.
Plasmin generation induces neutrophil aggregation: Dependence on the catalytic and lysine binding sites
Authors: Ryan Thomas J. , Lai Linda , Malik Asrar B.
Published: February 2005

We established that plasmin (10−10M to 10−6M) caused neutrophils (PMN) to aggregate using an in vitro assay. Plasminogen had no PMN aggregatory activity even at a concentration of 2 μM. However, plasminogen caused PMN to aggregate when incubated with plasminogen activators [tissue plasminogen activator (25–200 U/ml) or urokinase (5–500 U/ml)]. Tissue plasminogen activator and urokinase alone had no PMN aggregatory activity. Analysis of these incubation mixtures indicated that plasmin was generated in the process and that the time course of plasmin generation correlated with the aggregation response. Active‐site‐inhibited plasmin did not induce PMN aggregation, indicating that a functional catalytic site was required for the response. Pretreatment of PMN with either active‐site‐inhibited plasmin or tranexamic acid prevented PMN aggregation by plasmin, indicating that both binding of plasmin to the cell surface via the lysine binding sites and catalysis were required for the response. The generation of plasmin during activation of fibrinolysis may play a pro‐inflammatory role by mediating aggregation of PMN. © 1992 Wiley‐Liss, Inc.
Partial purification and characterisation of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy
Authors: Woodman Anthony C. , Selden Clare A. , Hodgson Humphrey J. F.
Published: February 2005

We have investigated the influences that nonparenchymal cells from regenerating rat liver exert on hepatocyte proliferation. When primary adult rat hepatocytes isolated from resting liver were co‐cultured with nonparenchymal cells (NPCs) from resting liver of a different syngeneic animal, the proliferative response of hepatocytes to epidermal growth factor (EGF) was unaffected by the presence of NPCs. In the presence of NPCs taken from livers that had undergone partial hepatectomy 24 hours before (regen‐NPCs), the response of hepatocytes from resting liver to EGF, TGF‐α, and hepatocyte growth factor (HGF) was markedly inhibited. Inhibitory activity was not dependent on cell‐to‐cell contact, and conditioned‐medium from regen‐NPCs, but not normal NPCs, inhibited EGF‐induced hepatocyte DNA synthesis by approximately 50%. After concentration by gel chromatography and lyophilisation, inhibition was 98%. The inhibitory activity migrated on SDS‐PAGE gel electrophoresis with an apparent molecular weight of 14 to 17 kDa and was trypsin‐sensitive but relatively heat‐stable. The effects of blocking antibodies established that it was not TGF‐β1, IL 1‐β, or IL 6. Investigations of regen‐NPCs taken at different time points demonstrated that inhibitory activity was released into conditioned medium of cells harvested at 24 and 48 hours after partial hepatectomy, but not 10 or 72 hours. This powerful inhibitor of hepatocyte response to proliferogens is released by cultures of NPCs with a time course suggesting that it may be involved in terminating the surge of hepatocyte replication induced by partial hepatectomy. © 1992 Wiley‐Liss, Inc.
Regulation of programmed death in erythroid progenitor cells by erythropoietin: Effects of calcium and of protein and RNA syntheses
Authors: Kelley Linda L , Koury Mark J. , Bondurant Maurice C.
Published: February 2005

Erythropoietin (EPO) retards DNA breakdown characteristic of programmed cell death (apoptosis) and promotes survival in erythroid progenitor cells. The mechanism by which EPO inhibits programmed death is unknown. In the well‐characterized model of glucocorticoid‐treated thymocytes, activation of a Ca2+/Mg2+‐sensitive endonuclease and new protein and RNA syntheses have been found necessary for apoptosis. We examined the effects of EPO on the free intracellular calcium ion concentration ([Ca2+]i), and the roles of Ca2+ and RNA and protein syntheses on DNA cleavage in erythroid progenitor cells. The murine model of erythroid differentiation using Friend leukemia virus‐infected proerythroblasts (FVA cells) was used. EPO did not affect the [Ca2+]i in FVA cells. Decreasing [Ca2+]i by extracellular Ca2+ chelation with EGTA facilitated DNA breakdown. Increasing [Ca2+]i with the calcium ionophore 4‐bromo‐A23187 increased DNA cleavage; however, DNA fragments generated by high [Ca2+]i were much larger than those seen in the absence of EPO or presence of EGTA. Increased [Ca2+]i also inhibited DNA breakdown to small oligonucleosomal fragments characteristic of cells cultured without EPO. However no concentration of ionophore protected the high molecular weight DNA as did EPO. Cycloheximide inhibited DNA breakdown in a dose dependent manner in cultures lacking EPO, but two other protein synthesis inhibitors, pactamycin and puromycin, did not prevent DNA breakdown. Inhibition of RNA synthesis with actinomycin D did not prevent DNA breakdown. Cells with morphological characteristics similar to those reported in other cells undergoing programmed death accumulated in EPO‐deprived cultures. These studies demonstrate that although DNA cleavage and morphological changes are common to apoptotic cells, the roles for Ca2+ and protein and RNA syntheses are not universal and suggest that apoptosis can be regulated by different biochemical mechanisms in different cell types. © 1992 Wiley‐Liss, Inc.
Cell density dependent effects of TGF‐β demonstrated by a plasminogen activator‐based assay for TGF‐β
Authors: Flaumenhaft Robert , Rifkin Daniel B.
Published: February 2005

Transforming growth factor‐β1 (TGF‐β1) induces a decrease in plasminogen activator (PA) expression in confluent cultures of bovine aortic endothelial (BAE) cells. We describe an assay using the suppression of PA expression in confluent BAE cells by TGF‐β1 which detects concentrations of the growth factor ranging from 5 to 200 pg/ml and has an ED50 of 15–20 pg/ml. The assay can be performed in 96‐well plates and requires a minimum of 35 ul of solution per sample, thereby limiting the amount of reagents required and allowing many samples to be tested in a single assay. Here we demonstrate that the effect of TGF‐β1 on PA expression in BAE cells depends on the length of time the cells are exposed to the growth factor and the density at which the cells are plated. In cells plated at a high density (3.5 × 105 cells/cm2), both 4 h and 24 h exposures to TGF‐β1 suppress PA expression. However, with cells plated sparsely (3.5 × 104 cells/cm2), a 4 h exposure to TGF‐β1 increases PA expression 2‐fold, whereas a 24 h exposure results in an 85% inhibition of basal PA expression. The paradoxical stimulation of PA expression in cells at a sparse density upon 4 h exposure to TGF‐β1 occurs in a dose‐dependent manner with an ED50 of 15–20 pg/ml. This bifunctional response of PA production in cells exposed to TGF‐β1 may have implications with regard to the role of TGF‐β1 in angiogenesis. © 1992 Wiley‐Liss, Inc.
Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: Role of epidermal growth factor and hormones
Authors: De Juan Carmen , Benito Manuel , Fabregat Isabel
Published: February 2005

Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non‐treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with this growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene. © 1992 Wiley‐Liss, Inc.
The proto‐oncogene C‐myc is involved in cell differentiation as well as cell proliferation: Studies on growth plate chondrocytes in situ
Authors: Farquharson Colin , Hesketh John E. , Loveridge Nigel
Published: February 2005

A combination of immunocytochemistry and microdensitometry has been used to localize and quantify the expression of the proto‐oncogene c‐myc within chondrocytes of the proximal growth plates of rat and chick long bones. Although the c‐myc protein was localized in all chondrocytes of the growth plate of both species the most intense staining was restricted to the proliferating and differentiating chondrocytes. These were identified by their ability to synthesize DNA (bromodeoxyuridine positive) and the presence of alkaline phosphatase activity, respectively. Species differences did exist with the c‐myc concentration of the chick proliferating and differentiating chondrocytes being higher (128% and 240%, respectively) than the respective chondrocytes of the rat. The higher c‐myc concentration in the chick proliferating chondrocytes paralleled the differences in the bromodeoxyuridine labelling index between the two species. In the rat, the concentration of c‐myc protein present in the differentiating chondrocytes was 74% higher than in the respective proliferating chondrocytes, while in the chick it was 146% higher. The data not only provides further evidence for a role of the c‐myc protein in cell proliferation but also suggests involvement of this protein in chondrocyte differentiation and/or hypertrophy. © 1992 Wiley‐Liss, Inc.
Induction of fibronectin gene expression by transforming growth factor beta‐1 is attenuated in bronchial epithelial cells by ADP‐ribosyltransferase inhibitors
Authors: Beckmann Joe D. , Illig Mary , Romberger Debra , Rennard Stephen I.
Published: February 2005

Transforming growth factor‐β (TGF‐β) exerts several effects on cultured airway epithelial cells including inhibition of proliferation and stimulation of fibronectin gene expression. ADP‐ribosylation is one potential regulatory mechanism of gene expression by TGF‐β. We tested this possibility by exposing cultured bovine bronchial epithelial cells to the chemical inhibitor of ADP‐ribosyl transferase enzymes, 3‐aminobenzamide (3‐AB) and, for comparison, 3‐aminobenzoic acid (3‐ABA), which is structurally similar to 3‐AB but which does not inhibit ADP‐ribosyl transferases. Exponential cell growth rate (1.2 doublings/day) or cellular morphology observed by phase contrast microscopy were not affected by 3 mM 3‐AB or 3‐ABA. Neither compound antagonized inhibition of cell division or induction of squamous morphology by TGF‐β1. In contrast, the sixfold stimulation of fibronectin production by exposure of cells to 30 pM TGF‐β1 for 48 h was reduced by 50% in the presence of 3 mM 3‐AB, whereas 3 mM 3‐ABA had no effect. The antagonistic effect was augmented by administration of 3‐AB 24 h prior to induction by TGF‐β1. Northern blot hybridization analyses demonstrated that 3‐AB, but not 3‐ABA, attenuated the induction of fibronectin mRNA by TGF‐β1 by up to 50%. These observations may implicate a role of cellular ADP‐ribosylation in the regulation of some gene expression by TGF‐β. © 1992 Wiley‐Liss, Inc.
Modulation of growth factor responsiveness of murine mammary carcinoma cells by cell matrix interactions: Correlation of cell proliferation and spreading
Authors: Elliott Bruce , Östman Arne , Westermark Bengt , Rubin Kristofer
Published: February 2005

We have examined the role of growth factors and extracellular matrix in the proliferation and cell adhesion of a murine mammary carcinoma, SP1, and a stable highly metastatic variant, SP1‐3M. On fibronectin, both cell types proliferated strongly in response to basic fibroblast growth factor (bFGF) and platelet‐derived growth factor BB (PDGF‐BB) after culture for 24 h and 72 h. In contrast, on collagen type I, SP1 cells proliferated only weakly to PDGF‐BB at either time, and SP1‐3M cells showed a response to PDGF‐BB only at 72 h. The proliferative response to bFGF was also consistently lower when the cells were cultured on collagen than on fibronectin. No significant proliferative responses were detected to epithelial growth factor (EGF), transforming growth factor‐β (TGF‐β), or estrogen on any substratum. The lack of responsiveness to PDGF‐BB of cells cultured on collagen type I was not due to differences in numbers or affinity of PDGF receptors. We therefore examined the adhesion and spreading properties of SP1 and SP1‐3M cells. Without exogenous growth factors, both cell lines adhered to fibronectin and laminin. SP1‐3M cells did not bind to collagen type I, whereas SP1 cells did. Attachment to all three substrata was inhibited by anti‐β1 integrin IgG, suggesting that the primary adhesion to these substrata is mediated by β1 integrins. SP1 and SP1‐3M cells showed similar integrin patterns following immunoprecipitation by anti‐β1 integrin IgG. bFGF stimulated increased adhesion and spreading of both SP1 and SP1‐3M cells to collagen type I within 24 h, whereas PDGF‐BB was less capable of this effect. Our results suggest that the proliferative response of SP1 and SP1‐3M cells to PDGF‐BB and bFGF is dependent on the extracellular matrix environment, and imply that modification of extracellular matrix and/or surface integrin receptors may regulate responsiveness to these growth factors in the SP1 tumor model. © 1992 Wiley‐Liss, Inc.
Effect of protein and steroidal osteotropic agents on differentiation and epidermal growth factor‐mediated growth of the CFK1 osseous cell line
Authors: Bernier Suzanne M. , Goltzman David
Published: February 2005

The effects of factors known to influence bone metabolism were examined using the osseous cell line CFK1. Parathyroid hormone (PTH) and dexamethasone (DEX) appeared to enhance the formation of cell foci of CFK1 cells in culture whereas retinoic acid (RA) caused a marked alteration in individual cell morphology. Bone morphogenetic protein (BMP‐2) and PTH increased alkaline phosphatase activity, however, this index of differentiation was suppressed by epidermal growth factor (EGF), DEX, and RA. BMP‐2 and EGF each stimulated DNA synthesis in a dose‐dependent manner and enhanced cell numbers, but, no synergistic response of EGF and BMP‐2 was observed. PTH and DEX failed to significantly alter cell number of EGF‐stimulated DNA synthesis or cell proliferation. Although RA treatment of CFK1 cells resulted in a reduction in cell number compared to control, pretreatment with RA enhanced EGF‐stimulated DNA synthesis and proliferative effects. At least part of this effect was by increasing the EGF receptor binding capacity of the cells. Furthermore, using cell cycle analysis, addition of EGF stimulated the progression of RA‐treated cells into the DNA synthesis (S) phase with a reduced lag time. EGF and BMP‐2, therefore, appear to exert a role in the expansion dynamics of the CFK1 population although BMP‐2 may also enhance differentiation. PTH and DEX may act primarily to modulate the differentiated function of the CFK1 cells. RA inhibited cell proliferation and may mediate differentiation towards a less established cell population with upregulation of EGF receptors. The CFK1 cell model may, therefore, provide insight into microenvironmental control of growth and differentiation of precursor osseous cells. © 1992 Wiley‐Liss, Inc.
UV induction of transforming growth factor alpha in melanoma cell lines is a posttranslational event
Authors: Chenevix‐Trench G. , Cullinan M. , Ellem K. A. O. , Hayward N. K.
Published: February 2005

Low, mitogenic fluences of UVC (3.7–5.6 Jm−2) have previously been shown to cause increases of radioimmunoassayable transforming growth factor alpha (TGFα) in the medium and cells of cultures of melanocytes, melanoma lines, and HeLa cells (Ellem, K.A.O., Cullinan, M., Baumann, K.C., Dunstan, A.: Carcinogenesis 9:797–801, 1988). Here the cellular mechanism of this increase is explored by Northern blotting to detect any changes in TGFα mRNA levels, and the use of inhibitors of macromolecular synthesis to attempt to block the increase in TGFα protein. We were unable to detect any increase in TGFα mRNA levels attributable to UVC between 2 and 24 hours after irradiation. Inhibition of DNA synthesis (arabinosylcytosine, 10 μM), RNA synthesis (actinomycin D, 3 μg/ml; DRB 93 μM), or protein synthesis (cycloheximide, 10 μg/ml) failed to prevent the UVC induced increase in TGFα. We conclude that the UVC induction of TGFα is by a posttranslational mechanism. There was considerable discordance between the amount of TGFα protein and its mRNA in cultures of 15 different melanoma cell lines, which again emphasized that posttranscriptional mechanisms modulate the release of immunodetectable TGFα. We also found that the inhibitors themselves were capable of inducing an increase in TGFα in MM229 cultures. This suggests that the inhibitors and UV may effect the increase by a common mechanism, perhaps the activation of cell surface proteases as suggested for other stimuli (e.g., Pandiella, A., and Massagué, J.: Proc. Natl. Acad. Sci., USA 88:1726–1730, 1991) and that the response may be part of a global response to perturbation of DNA synthesis. © 1992 Wiley‐Liss, Inc.
Age‐related alterations in superoxide anion generation in mouse peritoneal macrophages studied by repeated stimulations and heat shock treatment
Authors: Lavie Lena , Weinreb Orly , Gershon David
Published: February 2005

The ability of thioglycollate‐elicited peritoneal macrophages (PM) from young and senescent mice to generate superoxide anions (O−2) under repeated stimulation or thermal stress was studied using either zymosan, opsonized zymosan (OZ), or phorbol myristate acetate (PMA). A diminished capacity to recover from repeated stimulation was found with aging. When stimulated for a second time 24 hours after the primary stimulation, PM from young animals generated 80% of the initial O−2 responses to either zymosan, or OZ. Under the same conditions, PM from senescent mice generated 62% of the initial O−2 produced in response to zymosan, and 45% in response to OZ. In both age groups the response to a second PMA stimulation comprised only 10% of the primary response. A considerably diminished capacity to generate O−2 was also demonstrated in PM from senescent mice after recovery from exposure to thermal stress. Exposure to 42.5°C for 20 minutes was found to be the threshold temperature for irreversible loss of activity in senescent PM, whereas at this temperature, PM from young animals recovered up to 70% of their O−2 generating activity. Since NADPH oxidase and superoxide dismutase activities were only mildly affected by the hyperthermia in all age groups, they could not account for the age‐related decline in the recovery from stress. Age‐related alterations in signal transduction or receptor alterations could possibly play a primary role in this decline. © 1992 Wiley‐Liss, Inc.
Signal transduction during liver regeneration: Role of insulin and vasopressin
Authors: Marino M. , Mangiantini M. T. , Spagnuolo S. , Luly P. , Leoni S.
Published: February 2005

The relationship between cell proliferation and inositol lipid turnover has been studied by comparing the steady state of inositol derivative metabolism in quiescent and regenerating rat hepatocytes isolated at 4 h (G1 phase of first cell cycle) and 24 h (onset of M phase) after partial hepatectomy. The effect of two hormones able to regulate hepatic regeneration, insulin and vasopressin, has been considered, and the results can be summarized as follows: (i) at 4 h after partial hepatectomy, the precursor incorporation into inositol polyphosphates and the particulate phospholipase C activity increase with respect to quiescent hepatocytes, whereas the content of I1,4,5P3 does not change, suggesting an increased turnover of this molecule in this step of cell cycle priming; (ii) 24 h after partial hepatectomy, the radioactivity linked to IP3 and IP4, as well as soluble and particulate phospholipase C activity, and IP3 content increase, suggesting the presence, at the onset of M phase, of second messenger accumulation; (iii) only 24 h after partial hepatectomy, the inositol derivative metabolism is affected by vasopressin; and (iv) insulin exerts a modulatory role on inositol polyphosphate production without involving membrane‐bound PLC activity or phosphoinositide hydrolysis. These data suggest that inositol‐derived signal molecules are associated with hepatic regneration; moreover, the metabolic pathway of such compounds seems to be regulated so that only specific inositol phosphates are present in each step of the cell cycle. © 1992 Wiley‐Liss, Inc.
Role of protein kinase C in transforming growth factor‐β1 induction of carcinoembryonic antigen in human colon carcinoma cells
Authors: Chakrabarty Subhas
Published: February 2005

Transforming growth factor‐β1 (TGF‐β1) regulates the expression of the carcinoembryonic antigen (CEA) gene family in the human colon carcinoma cell line Moser. The mechanisms through which it acts, however, are unknown. In this communication, several lines of evidence are presented to show that the induction of CEA expression and secretion (collectively called CEA responses) by TGF‐β1 is associated with protein kinase C (PKC) pathway of signal transduction. Treatment of intact cells with the PKC‐specific inhibitor calphostin C downmodulated cellular PKC phosphotransferase activity and blocked the induction of the CEA responses by TGF‐β1. Depletion of PKC by treatment of intact cells with phorbol ester also blocked the action of TGF‐β1. The induction of the CEA responses by TGF‐β1 was also blocked by the protein kinase inhibitor 1‐(isoquinolinesulfonyl)‐2‐methylpiperazine dihydrochloride (H‐7), which also inhibited cellular PKC activity. However, TGF‐β1 did induce the CEA responses in intact cells treated with the calmodulin antagonist N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide hydrochloride (W‐7), the calmodulin‐dependent phosphodiesterase inhibitor calmidazolium, the diacylglycerol kinase inhibitor R59 022, and the G‐protein inhibitors cholera toxin and pertussis toxin. Treatment of intact cells with TGF‐β1 induced a rapid and transient increase in PKC phosphotransferase activity. TGF‐β1, however, was unable to induce PKC enzymatic activity in cells pretreated with calphostin C. Therefore, it is concluded that TGF‐β1 regulates the CEA responses through a signal transducing pathway associated with PKC. © 1992 Wiley‐Liss, Inc.
Transforming growth factor‐β1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells
Authors: Takenaka Ivone M. , Hightower Lawrence E.
Published: February 2005

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor‐β1 (TGFβ), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event, (3) TGFβ is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of Hsp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGFβ can substitute for serum in stimulating synthesis of members of the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGFβ on the hsp's. Analysis of the induction of hsp's using one‐and two‐dimensional polyacrylamide gel electrophoresis indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGFβ, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesis increased more slowly, undergoing an ∼twofold increase in 24 hours. Using a modified protocol for two‐dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90‐2 and ‐4). These phosphorylated isoforms turned over faster than the unphosphorylated forms of Hsp90. All four isoforms were heat inducible, but only Hsp90‐2 and ‐3 were induced rapidly by TGFβ, again within 5 hours of treatment. The effects of serum on these protein families were similar to those of TGFβ, suggesting that this cytokine may be the serum component primarily responsible for up‐regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase their chaperoning capacity for newly synthesized polypeptides in preparation for an increase in the rate of synthesis of proteins up‐regulated by TGFβ. © 1992 Wiley‐Liss, Inc.
Immunocytochemical localization of annexin V (CaBP33), a Ca2+‐dependent phospholipid‐and membrane‐binding protein, in the rat nervous system and skeletal muscles and in the porcine heart
Authors: Spreca Antonio , Rambotti Maria Grazia , Giambanco Ileana , Pula Grazia , Bianchi Roberta , Ceccarelli Paolo , Donato Rosario
Published: February 2005

We investigated the ultrastructural localization of annexin V a Ca2+‐dependent phospholipid‐ and membrane‐binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca2+‐dependent binding of the protein to brain and heart membranes, and on existence of both EGTA‐ and Triton X‐100‐extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells. © 1992 Wiley‐Liss, Inc.
Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers
Authors: Stasek Jerome E. , Patterson Carolyn E. , Garcia Joe G. N.
Published: February 2005

Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non‐muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca2+‐phospholipid‐dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and α‐thrombin, a receptor‐mediated agonist known to increase endothelial cell permeability, both induced rapid, dose‐dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by γ‐[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist‐mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist‐induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (∼200% increase with 10−6 M PMA, ∼400% increase with 10−8 M α‐thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC‐mediated phosphorylation of two specific CSPs, the actin‐ and calmodulin‐binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]‐labeled BPAEC monolayers stimulated with either PMA or α‐thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented α‐thrombin‐ and PMA‐induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist‐induced barrier dysfunction. These results demonstrate that agonist‐stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayers, an event which occurs in concert with agonist‐mediated endothelial cell contraction and resultant barrier dysfunction. © 1992 Wiley‐Liss, Inc.
Heat induced protein denaturation in the particulate fraction of hela S3 cells: Effect of thermotolerance
Authors: Burgman P. W. J. J. , Konings A. W. T.
Published: February 2005

In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the ‐SH groups of the proteins were labelled with a maleimido bound spin label (4‐maleimido‐tempo). The sample was heated inside the ESR spectrometer at a rate of 1°/min. ESR spectra were made every 2–3° between 20° and 70°. In the PF of untreated cells conformational changes in proteins were observed in three temperature streches: between 38 and 44° (transition A, TA); between 47 and 53° (transition B, TB); and above 58° (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat‐induced) thermotolerant ceils, TA was unchanged while TB and TC started at lated with the degree of thermotolerance that was induce in the cells. These results suggest that protection against heat‐induced denaturation of proteins in the PF in involved in heat induced thermotolerance. © 1992 Wiley‐Liss, Inc © 1992 Wiley‐Liss, Inc.
Basic fibroblast growth factor increases junctional communication and connexin 43 expression in microvascular endothelial cells
Authors: Pepper M. S. , Meda P.
Published: February 2005

We have analyzed the effect of basic fibroblast growth factor (bFGF) on junctional communication (coupling) and connexin 43 (Cx43) expression in bovine microvascular endothelial (BME) cells. In control confluent culturers, the incidence of coupling, as assessed by the intercellular transfer of microinjected Lucifer Yellow, was limited to 13% of injected cells, and decreased to 0% with time in culture. After exposure to bFGF (3ng/ml), the incidence of coupling was increased in a time‐dependent manner, reaching a maximum of 38% of microinjected cells after 10–12 hours. The extent of coupling, as assessed by scrape loading, was maximally increased 2.1‐fold 8–9 hours after addition of bFGF. bFGF also induced a 2‐fold increase in Cx43 as assessed by Western blotting and increased Cx43 immunolabelling at contacting interfaces of adjacent BME cells. Cx43 mRNA was likewise increased after exposure to bFGF in a time‐and dose‐dependent manner, with a maximal 6–7 fold increase after a 4 hour exposure to 3–10ng/ml. Finally, the increase in coupling and Cx43 mRNA expression observed after mechanically wounding a confluent monolayer of BME cells was markedly reduced by antibodies to bFGF, which have previously been shown to inhibit migration. Taken together, these results indicate that exogenous and endogenous bFGF increase intercellular communication and Cx43 expression in microvascular endothelial cells. We propose that the bFGF‐mediated increase in coupling is necessary for the coordination of endothelial cells during angiogenesis and other vessel wall functions. © 1992 Wiley‐Liss, Inc.
Methylation of atypical protein aspartyl residues during the stress response of HeLa cells
Authors: Ladino Cynthia A. , O'Connor Clare M.
Published: February 2005

A protein carboxyl methyltransferase (PCMT), which specifically modifies atypical protein L‐isoaspartyl and D‐aspartyl residues, is widely distributed in eucaryotic cells, but the factors that regulate its activity in vivo have not been identified. It has been proposed that the PCMT initiates the repair of structurally damaged proteins. To test the possibility that the concentration of structurally abnormal cellular proteins affects PCMT activity, protein carboxyl methylation reactions were studied in HeLa cells exposed to various stresses that increase the extent of protein unfolding in cells. Protein carboxyl methylation rates increased 70–80% during incubations at 42°C and remained elevated for periods of up to 8 hr. This sustained increase was greater than that predicted from thermal effects on the enzyme alone and may relect the exposure of atypical aspartyl sites as proteins unfold as well as increased rates of protein deamidation and isomerization at elevated temperatures. Methylation rates showed no increases following 12 hr incubations with the amino acid analogs L‐azetidine‐2‐carboxylic acid or L‐canavanine. Northern blot analysis of RNA preparations from control and stressed cells revealed three major transcripts for the PCMT in HeLa cells, which are 1.6, 2.6, and 4.5 kb in length. The concentrations of all three transcripts decreased by ∼ 20% from control levels during heat shock. No changes in PCMT transcript concentrations were observed during incubation with the amino acid analogs. By contrast, large increases in the concentrations of hsp70 and ubiquitin trascripts were observed following either heat or chemical stresses. The results demonstrate that the PCMT is a constitutive component of cells whose function is required under normal conditions as well as during stress conditions, which accelerate structural damage to cellular proteins. © 1992 Wiley‐Liss, Inc.
Constitutive HSP70: Oligomerization and its dependence on ATP binding
Authors: Kim Dooha , Lee Yong J. , Corry Peter M.
Published: February 2005

The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS‐polyacrylamide gel electrophoresis, showed multiple bands in native‐polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS‐polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1–10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [γ‐32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins. © 1992 Wiley‐Liss, Inc.
Ligand‐induced protein tyrosine kinase activity in living cells coexpressing intact EGF receptors and receptors with an extensive cytosolic deletion
Authors: Dou Yi , Hoffman Patricia , Hoffman Brian L. , Carlin Cathleen
Published: February 2005

A population of stable NIH 3T3 transfectants with two molecular weight classes of membrane‐bound EGF receptors encoded by a human EGF receptor cDNA has been identified and characterized. In addition to intact EGF receptors, these cells also express a molecule with an extensive cytosolic deletion. This deletion includes the ligand‐activated intrinsic protein tyrosine kinase catalytic domain. Treatment with EGF caused dimerization of intact and truncated receptors, allowing us to assess protein tyrosine kinase activity in the heterodimer isolated from living cells. In contrast to homodimeric complexes with intact EGF receptor only, heterodimers were deficient in protein tyrosine kinase activity. Moreover, physical association between intact and truncated molecules suppressed receptor autophosphorylation by EGF receptor protein tyrosine kinase activated by antibody binding in vitro. Evidence presented here supports the idea that protein tyrosine kinase activation is facilitated by interaction between adjacent receptor molecules with intact catalytic domains. Furthermore, molecules with cytoplasmic deletions that are physically associated with kinase‐active EGF receptors appear to behave as dominant negative mutations. The HerC cl cells used in this study were selected with methotrexate to amplify the EGF receptor cDNA, and in that sense may resemble certain tumor‐derived cells characterized by overexpressed and rearranged EGF receptor genes. © 1992 Wiley‐Liss, Inc.
Localization of p35 (annexin I, lipocortin I) in normal adult rat kidney and during recovery from ischemia
Authors: McKanna James A. , Chuncharunee Aporn , Munger Karen A. , Breyer Julia A. , Cohen Stanley , Harris Raymond C.
Published: February 2005

The 35‐kDa protein (p35, lipocortin I, annexin I), originally discovered as a Ca++‐dependent substrate for the EGF receptor tyrosine kinase, binds Ca++ and phospholipids, is developmentally regulated in embryos and has restricted expression in adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched in epithelia of Bowman's capsule, the macula densa, and medullary/papillary collecting ducts, suggesting that p35 is related to specialized renal functions. Light staining is observed in the thick ascending limb; elsewhere, immunoreactivity is nil. Since renal recovery from ischemia involves both hyperplasia and hypertrophy and reportedly is accelerated by EGF, we examined p35 distribution during this process. After 48 hours of recovery, both the distribution and amount of renal p35 are altered. Immunoblots show p35 levels increased at least threefold in whole‐kidney homogenates. The expression of p35 is still highly restricted in recovering kidneys; however, the thick ascending limb now stains heavily. This is the first documentation of alterations in annexin levels during a pathophysiologic response. However, our attempts to discern effects of exogenous EGF on the recovery from ischemia were negative for both mitotic index and renal function assays. © 1992 Wiley‐Liss, Inc.
Isolation of rat bone marrow mast lineage cells using Thy 1.1 and rat stem cell factor
Authors: Medlock Eugene S. , Mineo Chrysa , Housman Jerry M. , Elliott Gary S. , Trebasky Lisa D. , Langley Keith E. , Zsebo Krisztina M.
Published: February 2005

Recent reports have shown that various marrow‐derived cell populations respond vigorously to recombinant rat stem cell factor (rrSCF164), one form of the kitligand. In the present study, we isolated cell populations from rat bone marrow using the Thy 1.1 antigen (an antigen that in the rat is differentially expressed on primitive hemopoietic progenitor cells) and fluorescently conjugated rrSCF164 (rrSCF164‐PE). We show that rrSCF164 only stimulates cells that are enriched in the brightest Thy 1.1 populations (Thy 1.1bright). Numerous cell lines were generated by serial passage in rrSCF164 containing medium, and the prototypic cell lines have been designated SRT002 and SRT003. Each cell line retains the Thy 1.1bright phenotype and does not respond to interleukins (IL) 1–8, IL‐10, granulocyte (G) colony‐stimulating factor (CSF), granulocyte macrophage (GM) CSF, M‐CSF, or crude preparations of mitogen‐stimulated T‐cell supernatants. The Thy 1.1bright population of rat marrow was subdivided into a subset that binds rrSCF164‐PE (Thy 1.1bright, rrSCF164+). The majority of these cells possess certain characteristics in common with marrow‐derived mast cells and the Thy 1.1bright, rrSCF164 responsive cell lines, having similar granule morphology, being metachromatic, and reacting positively with alcian blue. Moreover, rats treated with rrSCF164 displayed significant increases in Thy 1.1bright, rrSCF164+ cells in the bone marrow. These studies show that the combination of Thy 1.1 and rrSCF164 makes possible the isolation of a unique subset of rat bone marrow cells that differentially express the Thy 1.1 antigen and the cell surface receptor c‐kit, the majority of which are morphologically similar to marrow‐derived mast cells. © 1992 Wiley‐Liss, Inc.
Cycloheximide protection against actinomycin D cytotoxicity
Authors: Borrelli Michael J. , Stafford Diane M. , Rausch Cynthia M. , Ofenstein John P. , Cosenza Steven C. , Soprano Kenneth J.
Published: February 2005

Pretreatment plus concomitant treatment with 10 μg/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c‐myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c‐myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c‐myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide‐induced reduction of actinomycin D bound to the acid precipitable fraction of the cells. © 1992 Wiley‐Liss, Inc.