摘要:

AbstractSickle cells contain internal vesicles which accumulate Ca2+. As shown here, the membrane enclosing the vesicles contains the plasma membrane Ca2+‐ATPase, or Ca2+pump, as judged by staining with an antibody directed against the protein. Moreover, the number of cells containing such vesicles increases upon deoxygenation. These findings argue strongly that the vesicles arise by endocytosis from the plasma membrane, and explain how they accumulate Ca2+. When sickle cells are depleted of ATP Ca2+is lost from the vesicles, as judged by the disappearance of staining with the Ca2+/membrane probe chlortetracycline (CTC), without a corresponding loss of antibody staining. This loss of Ca2+can be inhibited by nitrendipine, a Ca2+channel blocker. These results suggest that the vesicle membrane allows outward passage of Ca2+by a nitrendipine‐sensitive pathway, which can be overcome by the inward‐directed activity of the Ca2+pump of the vesicle membrane. If so, the Ca2+which vesicles contain is in dynamic equilibrium with the cytoplasm of the sickle erythrocyte. © 1992 Wiley‐L

ISSN:0021-9541    

DOI:10.1002/jcp.1041520102

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe NF‐κB family of transcription proteins represents multiple DNA binding,relrelated polypeptides that contribute to regulation of genes involved in immune responsiveness and inflammation, as well as activation of the HIV long terminal repeat. In this study multiple NF‐κB related polypeptides ranging from 85 to 45 kDa were examined for their capacity to interact with the PRDII regulatory element of interferon β and were shown to possess distinct intrinsic DNA binding affinities for this NF‐κB site and form multiple DNA binding homo‐ and heterodimer complexes in co‐renaturation experiments. Furthermore, using DNA templates containing two copies of the PRDII domain linked to the rabbit β globin gene the purified polypeptides specifically stimulated NF‐κB dependent transcription in an in vitro reconstitution assay as heterodimers but not as p50 homodimers. These experiments emphasize the role of NF‐κB dimerization as a distinct level of transcriptional control that may permit functional diversification of a limited number of regulatory proteins. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041520103

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

年代:1992

数据来源: WILEY

摘要:

AbstractTo assess the roles of developmental factors in the regulation of sheep IGFBP production at the cellular level, we characterized and compared the IGFBPs released by fetal, postnatal, and maternal sheep skin fibroblasts in culture with those in fetal, postnatal, and maternal sheep plasma. Sheep fibroblasts produced seven IGFBPs: a 36.5–41 kDa protein induced in vitro by IGF‐I, likely representing oIGFBP‐3; a 28.5 kDa protein that reacted with antisera to human IGFBP‐2, likely representing oIGFBP‐2; 25 and 27 kDa proteins induced in fetal fibroblasts by IGF‐I; a 22 kDa protein that was inhibited by IGF‐I, likely representing oIGFBP‐4; and 21 and 23 kDa proteins labelled only by IGF‐II, suggesting their similarities to IGFBP‐6. The developmental pattern of IGFBP production by sheep fibroblasts in culture was similar in several respects to that observed in sheep plasma. For example, relative amounts of the 21, 22, and 28.5 kDa IGFBPs exceeded that of the 36.5–41 kDa protein in early fetal fibroblast conditioned media and in fetal plasma, while the relative concentrations of the 36.5–41 kDa protein increased markedly during the perinatal period. Sheep plasma differed, however, in two major respects from fibroblast conditioned media: First, fetal, and to a far lesser extent maternal, plasma contained a 200 kDa IGF‐II‐selective BP, likely to be the circulating form of the IGF‐II receptor; and second, plasma, unlike conditioned media, contained a 26 kDa IGFBP, likely to be oIGFBP‐1. The results of our studies suggest that the production and release of IGFBPs by isolated sheep fibroblasts is regulated by developmental factors operative under in vitro culture conditions. The differences in the relative levels of IGFBPs in conditioned media from fetal, postnatal, and maternal sheep fibroblasts resemble in several respects the differences in the relative concentrations of the various IGFBPs in fetal, postnatal, and maternal sheep plasma. Thus, sheep fibroblasts provide a useful though imperfect model system by which to examine the nutritional and hormonal regulation of sheep IGFBP production at various developmental

ISSN:0021-9541    

DOI:10.1002/jcp.1041520104

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH‐related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR‐106 which possessed PTH‐responsive dual signal transduction systems (cAMP‐dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amilorideinhibitable Na+/H+exchange system. Both human (h)PTH‐(1–34) and hPTHrP‐(1–34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose‐dependent manner with a minimal effective dose of 10−10M. Dibutyryl cAMP (10−4M) and Sp‐cAMPS (10−4M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10−6M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP‐and Sp‐cAMPS‐induced decrease in pHi but did not affect calcium ionophore‐induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore‐induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA‐inhibited Na+/H+exchange and increased cytosolic calcium‐induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/

ISSN:0021-9541    

DOI:10.1002/jcp.1041520105

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

年代:1992

数据来源: WILEY

摘要:

AbstractIn previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon achronicexposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC‐PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp‐like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane‐associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two‐stage carcinogenesis are discussed. © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041520106

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) 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 ED50of 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 × 105cells/cm2), both 4 h and 24 h exposures to TGF‐β1 suppress PA expression. However, with cells plated sparsely (3.5 × 104cells/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 ED50of 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 i

ISSN:0021-9541    

DOI:10.1002/jcp.1041520107

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe subcellular distribution of glucose transporters in rat hepatocytes and HepG2 cells was studied in the absence and in the presence of insulin. Glucose transporters were quantitated by measuring glucose‐sensitive cytochalasin B binding and by protein immunoblotting using isoform‐specific antibodies. Plasma membrane contamination into subcellular fractions was assessed by measuring distribution of 5′‐nucleotidase and cell surface carbohydrate label. In hepatocytes, GLUT‐2 occurred in a low‐density microsomal (LDM) fraction at a significant concentration, and as much as 15% of cellular GLUT‐2 was found intracellularly that cannot be accounted for by plasma membrane contamination. In HepG2 cells which express GLUT‐1 and GLUT‐2, the two isoforms showed distinct subcellular distribution patterns: GLUT‐2 was highly concentrated in LDM while very little GLUT‐1 was found in this fraction, indicating that a large portion of GLUT‐2 occurs in intracellular organelles. Insulin treatment did not change the subcellular distribution patterns of glucose transporters in both cell types. Our results suggest that rat hepatocytes and HepG2 cells possess an intracellular storage pool for GLUT‐2, but lack the insulin‐responsive glucose transporter translocation mechan

ISSN:0021-9541    

DOI:10.1002/jcp.1041520108

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

年代:1992

数据来源: WILEY

摘要:

AbstractUnlike resident peritoneal macrophages (RPM) or tumor necrosis factor α (TNFα)‐primed bone marrow‐derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNFα‐primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan‐induced production of superoxide by unprimed and TNFα‐primed BMM and RPM, in spite of substantial inhibition of PMA‐induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10−7M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan‐induced response a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt‐induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpreation, the co‐stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan‐induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide productio

ISSN:0021-9541    

DOI:10.1002/jcp.1041520109

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

年代:1992

数据来源: WILEY

摘要:

AbstractLeukemia inhibitory factor/differentiation‐stimulating factor (LIF/D‐factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast‐like cells. Local bone‐resorbing factors such as IL‐1, TNFα, and LPS strongly induced expression of LIF/D‐factor mRNA in both clonal MC3T3‐E1 cells and primary osteoblast‐like cells. Neither parathyroid hormone nor 1α,25‐dihydroxyvitamin D3stimulated expression of LIF/D‐factor mRNA. LIF/D‐factor per se did not stimulate expression of its own mRNA.Appreciable amounts of LIF/D‐factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D‐factor, IL‐1, and IL‐6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D‐factor produced by osteoblasts is in concert with other boneresorbing cytokines such as IL‐1 and IL‐6 involved in the bone resorption seen in the joints of RA patients.LIF/D‐factor specifically bound to MC3T3‐E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D‐factor dose‐dependently suppressed incorporation of [3H]thymidine into MC3T3‐E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D‐factor alone had no effect on enzyme activity. These results suggest that LIF/D‐factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation wit

ISSN:0021-9541    

DOI:10.1002/jcp.1041520110

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

年代:1992

数据来源: WILEY

摘要:

AbstractIn vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an α1‐receptor (α1AR) to a β2‐receptor (β2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time‐dependent changes in α1‐ and β2‐adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of α1AR and β2AR, and the steady state levels of α1BAR and β2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in α1AR density, and a 70% decrease in α1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol‐induced but not in glucagon‐ or forskolin‐induced cAMP accumulation, no significant change in β2AR density, and a twofold increase in β2AR mRNA levels. Exposure of cells to cycloheximide, 2 μM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced β2AR densities, while the decrease in α1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in α1BAR mRNA and increase in β2AR mRNA levels and corresponding inverse changes in the synthesis of α1BAR and β2AR which account, at least in part, for the rapid conversion from α1‐ to β2‐adrener

ISSN:0021-9541    

DOI:10.1002/jcp.1041520111

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

年代:1992

数据来源: WILEY