摘要:

AbstractExpression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells (Orlow et al:J. Cell. Physiol.,142:129–136, 1990). Here, we show that internal and external receptors for MSH are of identical molecular weights (50–53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co‐purified with a sub‐cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs (Bologniaet al:J. Invest. Derm., 92:651–656, 1989). Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomittant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsivene

ISSN:0021-9541    

DOI:10.1002/jcp.1041470102

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

年代:1991

数据来源: WILEY

摘要:

AbstractPrevious studies to examine the effects of thrombocytopenia on thrombopoiesis have generally utilized immune‐mediated platelet depletion. We have developed a nonimmune model to exclude the possibility that adverse immune‐mediated effects have been misinterpreted as the physiological response to stimulation of thrombopoiesis. Thrombopoiesis was examined in mice after induction of thrombocytopenia with a single injection of the nonimmunologic agent neuraminidase (Ndase). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 8, 12, 24, 48, 72, 96, and 120 hr after administration of Ndase. Eight to 48 hr after induction of acute, severe throm‐bocytopenia (mean platelet count0.01). MK ploidy was maximally altered from normal at 72 hr with increased 32N MK frequency (32.0%,P0.005). Morphologic and morphometric examination of MK at all time points did not reveal detectable changes from normal in cytoplasmic appearance or size, respectively. Therefore, we have demonstrated marked alterations of morphology and size of platelets, and of MK ploidy, using this nonimmunologic model. These studies further support our previous observations that megakaryocyte ploidy and platelet volume are independently regulated in response to depletion of the circulating platelet mass, and they show that these changes are not dependent upon the mechanism of thro

ISSN:0021-9541    

DOI:10.1002/jcp.1041470103

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe have confirmed the hypothesis that a mitotoxin resulting from the conjugation of basic fibroblast growth factor and saporin exerts its cytotoxic effect through specific interaction with the basic fibroblast growth factor (FGF) receptor. Accordingly, the mitotoxin stimulates tyrosine phosphorylation of the 90 kD substrate that characterizes the initial cellular response to basic FGF. Cross‐linking experiments show that radio‐labeled basic fibroblast growth factor‐saporin (FGF‐SAP) binds to the receptor. Suramin, an inhibitor of growth factor receptor binding, inhibits the cytotoxicity of basic FGF‐SAP. In a study of 4 different cell types, there is a decrease in the ED50of the mitotoxin as the receptor number per cell increases. We have verified the cytotoxicity of the mitotoxin in 3 different assay systems. As expected, it is effective in the inhibition of protein synthesis and DNA synthesis, as well as of cell count. Binding of basic FGF‐SAP which will result in cytotoxicity occurs very rapidly: 5 minutes of incubation of 10 nM basic FGF‐SAP with cells results in 80% inhibition of cell count. The in vitro data indicate that basic FGF‐SAP is a receptor specific and potent suicide antagonist of basic FGF. Its potential as an anti‐FGF for therapeutic and research uses in

ISSN:0021-9541    

DOI:10.1002/jcp.1041470104

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro.Although the recovery of transferrin binding activity in exosomes is at best ∼25–30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if125I‐labelled transferrin receptor is measured using an immunological approach. Degradation products of the transferrin receptor in the medium suggest that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75–80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using3H‐nitrobenzylthioinosine binding to measure the nucleoside transporter.These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling.We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red c

ISSN:0021-9541    

DOI:10.1002/jcp.1041470105

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe compared the breakdown of total cellular protein with that of the contractile protein, myosin, in cultured C2 mouse skeletal myotubes. The degradation of long‐lived cellular proteins (which comprise the vast majority of myotube proteins) was inhibited by serum, insulin, and rat insulin‐like growth factor‐2. A physiological concentration of insulin was effective, but most of the effect of insulin occurred at concentrations well above the physiological range. IGF‐2 inhibited protein breakdown at concentrations well within the range of total IGF‐2 known to be present in the serum of fetal and neonatal rats. The breakdown of short‐lived proteins was not altered by insulin or serum. We measured myosin degradation using a monoclonal antibody directed against myosin heavy chain. The half‐life of myosin was 27 hours, and myosin breakdown was not altered by withdrawal of serum. Therefore, the enhanced protein degradation in response to serum withdrawal applies to certain proteins, but

ISSN:0021-9541    

DOI:10.1002/jcp.1041470106

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

年代:1991

数据来源: WILEY

摘要:

AbstractInteractive regulation of gene expression by retinoic acid (RA) and cyclic adenosine monophosphate (cAMP) in mammary tumor cells was explored using Shionogi mouse mammary carcinoma cells (SC115) as a model and urokinase‐type plasminogen activator (uPA) as a target gene product. Twenty‐four hour treatment of SC115 cells with 100 nM RA, 1 mM 8‐bromo‐cAMP (BrcAMP), and 100 nM RA + 1 mM BrcAMP resulted in extracellular uPA activity increases of 1.4‐fold, sevenfold, and 20‐fold, respectively. These effects were dose‐dependent with regard to both interacting members. Similar responses were obtained if 1 nM cholera toxin or 10 μM forskolin was used instead of the cAMP analog. Retinoids lacking the carboxylic acid function were inactive. The changes in uPA activity were accompanied by similar changes in uPA antigen concentration, as seen via Western blot analysis, and uPA mRNA abundance, as seen via Northern blot analysis. Actinomycin D, an inhibitor of RNA synthesis, blocked uPA stimulation by BrcAMP, suggesting that mRNA levels were transcriptionally regulated. The effect of BrcAMP on extracellular uPA activity was first evident at 2 h and peaked at 6 h; the effect of RA alone and the synergistic response to joint treatment, however, followed a slower time course, requiring at least 12 h for initial expression and increasing gradually with time up to at least 48 h. Priming with RA for 48 h followed by extensive washing of the cells resulted in a threefold enhancement of the stimulatory effect of BrcAMP on uPA. Experiments utilizing the casein/plasminogen overlay method for the detection of uPA secretion by single cells showed that the enhanced response to BrcAMP was due to an increased rate of uPA secretion per cell rather than to an increased fraction of uPA‐secreting cells. Initial investigation of the mechanism of RA potentiation of cAMP responsiveness showed that RA did not alter cellular cAMP levels or total cAMP‐dependent protein kinase A activity. Finally, the tumor promoter phorbol myristate acetate, an activator of protein kinase C, also increased SC115 cell uPA activity and synergized with RA. This raised the possibility that the enhancement of cAMP responsiveness by RA was indirectly mediated via an effect on protein kinase C. Experiments with protein kinase C‐depleted cells, however, showed that this was not the case. In conclusion, RA treatment of SC115 cells potentiates the effect of cAMP on uPA expression at the single cell level via a partially irreversible mechanism independent of protein kinase C. The molecular target of RA and whether SC115 cell differentiation underlies the effect of RA rema

ISSN:0021-9541    

DOI:10.1002/jcp.1041470107

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

年代:1991

数据来源: WILEY

摘要:

AbstractMetabolic events stimulated by epinephrine and norepinephrine in hepatocytes isolated from fetal and early postnatal male rats are largely mediated through the β2‐adrenergic receptor‐/cyclic AMP dependent‐system, whereas the same stimuli are transduced through the α1‐adrenergic receptor‐/phosphatidylinositol dependent‐system in hepatocytes isolated from young adult male rats. This developmental transition was investigated by correlating hepatic α1‐ and β2‐adrenergic receptor gene transcript levels with receptor levels as determined with selective radioligands in livers from late fetal to postnatal day 120 male Sprague‐Dawley rats. β2‐Adrenergic receptor concentration, initially high in membrane preparations isolated from fetal livers (203 ± 21 fmol/mg protein), dropped precipitously n postnatal day 6 livers (14± 2 fmol/mg protein) and remained low throughout development out to postnatal day 90.β‐Adrenergic receptor mRNA levels were highest in fetal livers, were decreased somewhat in postnatal day 6 livers and were uncetectable in livers beyond postnatal day 15. In contrast, hepatic α‐adrenergic receptor concentration was relatively low in fetal livers (86 ± 25 fmol/mg protein) and remained low until postnatal day 18. Thereafter, a steady increase in α1‐adrenergic receptors was observed until adult levels. (270 ± 24 fmol/mg protein) were achieved at postnatal day 27. α1‐Adrenergic receptor mRNA levels increased ∼ 3‐fold, reaching a peak at postnatal day 24. Surprisingly, at postnatal day 30 hepatic α1‐adrenergic receptor mRNA levels dropped to fetal levels; but, gradually increased with continued development. Thus, hepatic α1‐ and β2‐adrenergic receptors appear to be under complex regulatory control which may include transcr

ISSN:0021-9541    

DOI:10.1002/jcp.1041470108

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

年代:1991

数据来源: WILEY

摘要:

AbstractLaminin is a potent stimulator ofneurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium‐proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108‐15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin‐induced neurites. A four‐fold increase in the number of branches per neurite and a two‐to‐three‐fold increase in the number of neurites per cell were observed in both NG108‐15 and PC12 cells cultured on laminin when either 8‐BrcAMP or forskolin was added. The cAMP‐induced branching was also observed when PC12 cells were cultured on a laminin‐derived synthetic peptide (PA22‐2), which contains the neurite‐promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22‐2 were axonal, not dendritic, and the cAMP‐induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin‐mediated neurite outgrowth, but cAMP can

ISSN:0021-9541    

DOI:10.1002/jcp.1041470109

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

年代:1991

数据来源: WILEY

摘要:

AbstractPrevious studies have shown that ATP enhances intracellular calcium concentration and activates potassium channels in Madin Darby canine kidney (MDCK)‐cells, thus leading to hyperpolarization of the cell membrane. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of ATP on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12‐O‐tetradecanoyl phorbol 13‐acetate diester (TPA). In untreated cells, ATP leads to a sustained hyperpolarization and an increase of inositol 1,4,5‐tris‐phosphate (IP3), inositol 1,3,4,5‐tetrakisphosphate (IP4), and Cai. In the absence of extracellular calcium, the effect of ATP on PD and Cai is only transient. In cells pretreated with pertussis toxin, the effect of ATP on inositol trisphosphate is almost abolished, but ATP still leads to an increase of PD and Cai, which is sustained in the presence, and transient in the absence, of extracellular calcium. In cells pretreated with TPA, the effect of ATP on inositol trisphosphate is reduced and the effect on Cai blunted; but ATP still leads to a hyperpolarization of the cell membrane, which is sustained in the presence, and transient in the absence, of extracellular calcium. The observations indicate that ATP activates phospholipase C by a phorbol ester and pertussis toxin sensitive mechanism. In addition, ATP enhances Cai by pertussis toxin insensitive mechanisms allowing recruitment of calcium from both, extracellular fluid and intracellular stores. Calcium then activates the potassium channels and thus leads to the hyperpolarization of the

ISSN:0021-9541    

DOI:10.1002/jcp.1041470110

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

年代:1991

数据来源: WILEY

摘要:

AbstractRecent studies suggest that the nature of events leading to the formation, maintenance, and elimination of synapses may be regulated by cascade‐type, locally expressed proteases and protease inhibitors acting on adhesive extracellular matrix components. We have identified a molecule in conditioned medium of murine skeletal muscle cells that in molecular weight, target protease inhibition, heparin‐binding and cross‐reactivity with authenic antisera is similar to the human serine proteinase inhibitor, protease nexin I. Protease nexin I is a 43–50 kDa glycoprotein of the serpin superfamily (arg‐serpin class). Purified anti‐protease nexin I antibody (anti‐47 kDa) stains adult mouse skeletal muscle in discrete foci that precisely superimpose on synaptic neuromuscular junctions. Protease nexin I appears in patches on surfaces of cultured mouse skeletal myotubes, but not on myoblasts. These patches co‐localize with acetylcholine receptor clusters and acetylcholinesterase staining during cellular maturation in culture. Evidence that protease nexin I is a synaptic, extracellular antigen is particularly intriguing since it has been shown to be identical, in structure and activity, with a factor released by glial cells, called glia‐derived nexin that stimulates mouse neuroblastoma cell neurite outgrowth and inhibits granule cell migration Protease nexin I inhibits both tumor cell and myoblast plasminogen activator‐mediated destruction of extracellular matrix. Thus, such observations as presented in this report provide further evidence for involvement of cascade proteolytic systems, and their post‐translational regulation by specific serpins, in the remodeling that occurs in synapse form

ISSN:0021-9541    

DOI:10.1002/jcp.1041470111

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

年代:1991

数据来源: WILEY