摘要:

AbstractEthanol reportedly is immunosuppressive, interfering with lymphocyte proliferation. To investigate the basis for this immunosuppression, the effects of acute treatment with ethanol were studied on Ca2+mobilization in tonsillar B lymphocytes and the human lymphoblastoid B‐cell line, Ramos. The level of intracellular Ca2+was monitored in cells loaded with the fluorescent dye indo‐1 following stimulation with either anti‐lgM antibody or platelet activating factor. The effect of ethanol was also examined on the induction of the early proto‐oncogene c‐fosin these cells. Ethanol inhibited ligand‐activated Ca2+mobilization due to transmembrane influx but not intracellular store release, in a dose‐and time‐dependent manner. This inhibition was not due to the inability of anti‐lgM to bind to its surface receptor nor to membrane depolarization induced by ethanol. Ethanol also inhibited the Ca2+‐dependent induction by anti‐lgM of c‐fosin these cells. The inhibitory effects of ethanol on ligand‐activated Ca2+channels and subsequent induction of c‐fosmay provide the basis for its immunosuppressive act

ISSN:0021-9541    

DOI:10.1002/jcp.1041520302

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

年代:1992

数据来源: WILEY

摘要:

AbstractProtein kinase C (PKC) was implicated as an important positive regulator of angiogenesis by studies showing that tumor promoting phorbol esters, which activate PKC, stimulate angiogenesis both in vitro and in vivo. Therefore, inhibitors of PKC might be expected to block angiogenesis. MDL 27032 [4‐propyl‐5‐(4‐pyridinyl)‐2(3H)‐oxazolone], an inhibitor of cellular protein kinases, prevented capillary‐like tube formation by human umbilical vein endothelial cells (HUVEC) on basement membrane preparations, an in vitro model for angiogenic activity. MDL 27032 had an IC50= 50 μM, whereas MDL 27044, the 4‐methyl analog of MDL 27032, was less effective (IC50>100 μM). This selectivity was reflected in the relative abilities of the two compounds to inhibit PKC and protein kinase A (PKA) activity prepared from HUVEC, and also to inhibit the basic fibroblast growth factor stimulated proliferation of HUVEC. MDL 27032 (0.3 μg/egg) also significantly inhibited neovascularization in yolk sac membranes of developing chick embryos, whereas MDL 27044 added at concentrations up to 3 μg/egg was not inhibitory when compared with vehicle treated controls. Adhesion of HUVEC to individual extracellular matrix proteins, including laminin, fibronectin, and fibrinogen, but not to the mixture of matrix components or collagen type I and IV, was inhibited after treatment with MDL 27032. These studies suggest that MDL 27032, may have potential as an anti‐angiogenic agent because it disrupts both formation of tube‐like structures by HUVEC on Matrigel and normal neovascularization in ovo. This inhibition may in part be due to altered cellular interactions with the extracellular matrix.

ISSN:0021-9541    

DOI:10.1002/jcp.1041520303

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophgy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4‐fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient‐deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vaculoes was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components. © 1992 Wiley‐Lis

ISSN:0021-9541    

DOI:10.1002/jcp.1041520304

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

年代:1992

数据来源: WILEY

摘要:

AbstractTo study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse‐chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCI extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCI extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time‐dependent loss in sulphate from the BSP was evident. Using a 24 h pulse‐labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12‐day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation. © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041520305

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe regulation of β1‐ and β2‐adrenergic receptors (β1AR and β2AR) and receptor gene expression by interleukin‐1α (IL‐1α) was studied in cultured A549 human lung adenocarcinoma cells. The density and affinity of β1AR and β2AR were analyzed by computerized curve fitting of125I‐pindolol binding and its displacement by subtype selective antagonists. Steady state levels of receptor mRNAs were quantified by DNA excess solution hybridization assays. A549 cells in preconfluent cultures had fewer β1AR than β2AR (β1: 1.9 ± 0.3 vs. β2: 4.0 ± 0.5 fmol/mg protein, means ± SE), but lost most of their β2AR upon reaching confluency (β1: 2.7 ± 0.4, β2: 0.8 ± 0.3 fmol/mg). Incubation of preconfluent cells for 24 hr with 20 pM of human recombinant IL‐1α did not modify the density of either of the βAR subtypes. Similar incubations of confluent cells increased the density of β2AR from 0.8 ± 0.3 to 4.2 ± 0.9 fmol/mg, while the density of β1AR and the antagonist affinities of both receptors remained unaltered. The IL‐1α‐induced increase in β2AR density in confluent cells was antagonized in a concentration‐dependent manner by a recombinant protein antagonist of type I IL‐1 receptors (IC50: 0.2 nM). The IL‐1α‐induced increase in β2AR density was preceded by an increase in the steady state level of β2AR mRNA, while levels of β1AR mRNA remained unchanged. IL‐1α increased the stability as well as the rate of transcription of β2AR mRNA. These findings demonstrate for the first time that activation of type I IL‐1 receptors in A549 cells leads to a cell density‐dependent, selective upregulation of β2AR, and that the mechanism of this effect involves increased for

ISSN:0021-9541    

DOI:10.1002/jcp.1041520306

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

年代:1992

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐β (TGF‐β) is a multifunctional polypeptide, abundant in bone, that regulates both proliferation and differentiation of a wide variety of cells, but its role in osteoclast differentiation remains controversial. We have recently shown that long‐term cultures of human cord blood monocytes, in the presence of 1,25 dihydroxycholecalciferol (1,25‐(OH)2D3), give rise to cells that express two markers of the osteoclast phenotype, namely, the vitronectin receptor (VNR) and the calcitonin receptor (CTR). TGF‐β enhanced the proportion of cells expressing the VNR.In the present study, we investigated the effect of TGF‐β on the expression of CTR in cord blood monocytes cultured during 3 weeks in the presence of 1,25‐(OH)2D3. When added within the first 2 weeks of culture, TGF‐β (500 pg/ml) significantly decreased the cell protein content. TGF‐β alone did not stimulate basal cAMP production. The 10 nM‐sCT‐stimulated cAMP production was enhanced by increasing TGF‐β concentrations from 50 pg/ml to 1,000 pg/ml: for 500 pg/ml TGF‐β, it was 294 ± 28% vs. 140 ± 25% for control cultures (p<0.01). The sCT dose‐response curves showed a higher cAMP production from 10−9M to 10−7M of sCT in the presence of 500 pg/ml TGF‐β than in control cultures. The increase was 325 ± 36% in the presence of TGF‐β and 195 ± 13% in the absence of TGF‐β, for 10−7M sCT (p<0.01). This effect of TGF‐β on cAMP production was not observed either when it was added to monocyte cultures the last day or 2 hours before the end of the culture or in MCF7, a human breast cancer cell line that expresses CTR. [125I]‐sCT binding studies performed on confluent cells showed similar Kdin control and TGF‐β‐treated cells. By contrast, the CTR number was significantly increased in the presence of TGF‐β: 6.1 ± 2 × 104receptors per cell in control cultures and 28.8 ± 8.1 × 104receptors per cell in TGF‐β‐treated cultures (p<0,05). It is thus suggested that TGF‐β increases the number of CTR of these cells that have other features of preosteoclasts. The role of this cytokine on the process of osteoclast di

ISSN:0021-9541    

DOI:10.1002/jcp.1041520307

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

年代:1992

数据来源: WILEY

摘要:

AbstractTransforming 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 transd

ISSN:0021-9541    

DOI:10.1002/jcp.1041520308

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

年代:1992

数据来源: WILEY

摘要:

AbstractThioglycollate‐elicited macrophages (mϕ), upon binding the lectinGriffonia simplicifolia IB4(GSIB4) at the plasma membrane, are induced to secrete several low molecular weight proteins. In this investigation, results from specific ELISA and immunoprecipitation analysis of these molecules confirmed that the cytokine, tumor necrosis factor‐α (TNF‐α), belongs to the group of elicited proteins. This specific mϕ response is directly influenced by the dose of GSIB4used and the time in contact with the cells. At 40 μg/ml GSIB4, the maximum dose of lectin used, the mϕ activity was equal to that achieved when the cells were incubated with an interferon‐γ/lipopolysaccaride (IFN/LPS) stimulus alone. Moreover, the data showed that TNF‐mediated tumoricidal activity was significantly influenced by GSIB4binding to the mε membrane. When the lectin was incubated alone or in sequence with IFN/LPS, this ligand‐receptor binding promoted the lysis of WEHI 164 tumor target cells. However, concurrent incubation of both IFN/LPS and GSIB4with mϕ significantly diminished the tumoricidal response. This suggested that one of the metabolic pathways utilized subsequent to receptor‐ligand binding was altered by these interactions. When cyclic AMP (cAMP) and inositol triphosphate (IP3) levels were examined, the results showed that the concentration of cAMP was unchanged despite the fact that IP3levels were significantly enhanced upon mϕ‐GSIB4binding. Collectively, the data show that GSIB4binding to specific glycoproteins in the mϕ membrane induces TNF‐α production and facilitates TNF‐α dependent tumoricidal responses. It also appears that the transduction of the signal, in part, at least utilizes the phosphatidyl inositol pathway. Finally, it is noteworthy that mϕ activity is influenced by the sequence in which GSIB4is presented to the mϕ relative to the IFN/LPS

ISSN:0021-9541    

DOI:10.1002/jcp.1041520309

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

年代:1992

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐β1 (TGF‐β1) is a potent growth inhibitor for many cell types. On fibroblasts, TGF‐β1 has been shown to inhibit human platelet‐derived growth factor (PDGF)‐induced mitogenicity. The mechanism implicated in this growth inhibition is unknown. In this work, we show on human bone marrow fibroblasts that TGF‐β1, which inhibited PDGF‐BB mitogenicity, was able to block PDGF‐BB‐induced early events such as polyphosphoinositide (Ptdlns 4,5‐P2, Ptdlns 4‐P, and Ptdlns) breakdown and Ins 1,4,5‐P3formation. No significant modification by TGF‐β1 of PDGF‐BB binding (n1= 200,000 vs. n2= 195,000 sites per cell with TGF‐β1; Kd1= Kd2= 0.5 × 10−9M) and of internalization kinetics was observed. In addition, TGF‐β1 was shown to inhibit PDGF‐BB receptor autophosphorylation either in intact cells or in partially isolated membranes and to partially inhibit PDGF‐R tyrosine kinase activity. Since a dephosphorylation mechanism through protein phosphatases could be implicated, we used okadaic acid, a potent inhibitor of type 1 and 2A serine/threonine phosphatases and showed that okadaic acid restored PDGF‐receptor autophosphorylation on tyrosine residues. Based on these data, we suggest that an alternative regulatory mechanism of PDGF tyrosine phosphorylation seems to involve serine/threonine

ISSN:0021-9541    

DOI:10.1002/jcp.1041520310

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe used the osteogenic sarcoma cell line, UMR‐106‐01, to determine whether the rise in free cytosolic Ca2+concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two × 10−7M of the PTH antagonist8,18Nle34Tyr‐bPTH(3–34) amide ([Nle, Tyr]bPTH(3–34)A) was required to inhibit 10−9M bPTH(1–34)‐stimulated cAMP generation by 50%. 10−7M bPTH(1–34) completely overcame the inhibition induced by 10−6M [Nle, Tyr]bPTH(3–34)A. Only 7 × 10−8M and 2.7 × 10−7M [Nle, Tyr]bPTH(3–34)A were required to half maximally inhibit the [Ca2+]iincrease evoked by 3 × 10−8and 10−7M bPTH(1–34), respectively. In addition, dissociation between [Ca2+]iand cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH‐evoked [Ca2+]iincrease. Short incubation with PGF2αaugmented the PTH‐evoked [Ca2+]iincrease. Similar pretreatments had no effect on the PTH‐stimulated cAMP increase. Finally, preincubation with 1.5 × 10−9M bPTH(1–34) for 20 minutes almost completely blocked the effect of 10−7M bPTH(1–34) on [Ca2+]i, while preincubation with 5 × 10−9M bPTH(1–34) for 4 hours was required to inhibit the effect of 10−8M bPTH(1–34) on cAMP production by 50%. The differences in the regulation of the two PTH‐stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the

ISSN:0021-9541    

DOI:10.1002/jcp.1041520311

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

年代:1992

数据来源: WILEY