Cell regulation最新文献

Vanadate, an inhibitor of phosphotyrosyl phosphatases that exerts insulin-like effects in intact cells, stimulated both maturation and glucose uptake in isolated Xenopus laevis oocytes. Vanadate enhanced the effects of insulin/IGF-I and progesterone on maturation in a dose-dependent manner, with an effective concentration of 750 microM and a maximum at 2 mM, whereas, in the absence of hormone, activation of maturation was seen at 10 mM vanadate. Further, vanadate at 2 mM increased glucose uptake, but this effect was not additive to that of the hormone. In cell-free systems, vanadate caused a 12-fold stimulation of autophosphorylation of the oocyte IGF-I receptor in the absence, but not in the presence, of IGF-I and inhibited largely, but not totally, receptor dephosphorylation induced by an extract of oocytes rich in phosphotyrosyl phosphatase activities. These effects were dose dependent, with effective concentrations of 50-100 microM and maxima at 2 mM. Moreover, using an acellular assay to study the effect of vanadate on the activation of maturation promoting factor (MPF), we found that vanadate at 2 mM stimulated the activation of the MPF H1 kinase. This suggests that vanadate did not prevent dephosphorylation of p34cdc2 on tyrosine residues. Vanadate thus exerted insulin-like effects in oocytes, including stimulation of maturation. These effects might result from a direct or indirect action of vanadate on the IGF-I receptor kinase and on MPF activity.

To examine the diversity of the cadherin family, we isolated cDNAs from brain and retina cDNA preparations with the aid of polymerase chain reaction. The products obtained included cDNAs for two of three known cadherins as well as eight distinct cDNAs, of which deduced amino acid sequences show significant similarity with the known cadherin sequences. Larger cDNA clones were isolated from human cDNA libraries for six of the eight new molecules. The deduced amino acid sequences show that the overall structure of these molecules is very similar to that of the known cadherins, indicating that these molecules are new members of the cadherin family. We have tentatively designated these cadherins as cadherin-4 through -11. The new molecules, with the exception of cadherin-4, exhibit features that distinguish them as a group from previously cloned cadherins; they may belong to a new subfamily of cadherins. Northern blot analysis showed that most of these cadherins are expressed mainly in brain, although some are expressed in other tissues as well. These findings show that the cadherin family of adhesion molecules is much larger than previously thought, and suggest that the new cadherins may play an important role in cell-cell interactions within the central nervous system.

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.

Transforming growth factor-beta 1 (TGF-beta 1) is encoded predominantly by a 2.4-kb mRNA in most tissues. However, an additional transcript of 1.9 kb can be detected in rat heart after experimental myocardial infarction caused by ligation of the left coronary artery. This transcript level is significantly higher in infarcted heart tissue than in normal heart tissue, suggesting an important role for this mRNA species in response to injury. Structural characterization of the 1.9-kb mRNA showed that it included the entire coding sequence present in the 2.4-kb TGF-beta 1 mRNA, but also contained an additional nonhomologous 3'-untranslated region (UTR). The junction between the shared and unique 3' sequence in the 1.9-kb mRNA occurred only two nucleotides before the proposed polyadenylation site of the rat TGF-beta 1 2.4-kb mRNA. The unique 3'-UTR and the deduced shortened 5'-UTR in the novel 1.9-kb TGF-beta 1 mRNA suggest different transcriptional and translational regulatory mechanisms under conditions of tissue injury.

An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.

Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.

Crosslinking of the IgE receptor on the surface of rat basophilic leukemia (RBL) cells by multivalent antigen induces an association of these receptors with the detergent-insoluble membrane skeleton. Detergent insolubility of the receptor can also be induced on purified plasma membranes isolated from RBL cells by the use of either IgE oligomers or IgE monomers plus multivalent antigen. The critical event in initiating this interaction between the receptor and the membrane skeleton is cross-linking of the receptor. This association is rapid, and, when triggered by multivalent antigen, it is quickly reversed by the addition of excess monovalent antigen. The fact that this association occurs with the use of purified plasma membranes indicates that all of the components necessary for this interaction are present in the plasma membrane and that intracellular components are not required. Although crosslinking of the receptor activates phospholipase C and phospholipase A2 leading to the generation of several second messengers, none of these signaling mechanisms appears to be involved in IgE receptor interaction with the membrane skeleton. This interaction cannot be induced by phorbol 12-myristate 13-acetate (PMA), ionomycin, or a combination of these two reagents, although this will result in degranulation. Furthermore, receptor detergent insolubility is temperature independent when triggered by multivalent antigen, thus indicating that enzyme-catalyzed reactions are not important. This was verified by the fact that a variety of inhibitors that block phosphatidylinositol metabolism, arachidonic acid metabolism, Ca2+ influx, and protein kinase C (PKC) activation had no effect on antigen-induced association of the receptor with the membrane skeleton. These results indicate that the signaling mechanisms leading to the degranulation response are not involved in the association of the crosslinked receptor with the membrane skeleton.

Egr-1 is an "immediate early" gene that is induced by growth factors and agents that induce differentiation and encodes a protein with a "zinc-finger" motif. This protein is believed to be involved in transcriptional regulation. Because the fate of the kidney, and hence the organism, after an ischemic insult is dependent upon cellular repair, differentiation, and proliferation, we examined whether there was expression of the Egr-1 protein after an ischemic insult to the rat kidney. We have previously reported that Egr-1 mRNA accumulates to high levels in mouse kidneys after 30 min of ischemia and 1 h of reperfusion. In the present study, performed in rats, we show that Egr-1 mRNA transiently accumulates to very high levels after 40 min of ischemia and 1 h of reperfusion, is decreased by 3 h, and is nondetectable by 24 h of reperfusion. Reperfusion is required for Egr-1 protein accumulation to occur. The Egr-1 protein was localized by immunohistochemical techniques primarily to the nuclei of the thick ascending limbs and principal cells of the collecting ducts in the cortex and medulla. The subcellular localization was exclusively nuclear. There was some staining of the glomerular tuft and staining was particularly prominent in the parietal epithelial cells. In parallel to the accumulation of Egr-1 mRNA, the expression of the protein was transient and was no longer apparent after 5 h of reperfusion. The Egr-1 protein may play an important role in regulation of the response to ischemia of those segments of the nephron that are highly susceptible to oxygen deprivation and have a high level of intrinsic plasticity. It is possible that this protein may modulate cellular processes important for the ultimate ability of these critical nephron segments to recover from an ischemic insult.

An egg-specific NADase has been purified from the ovotestis of the marine mollusk Aplysia californica. The enzyme converts NAD to cyclic ADP-ribose (cADPR), which is a potent mobilizer of Ca2+. It is likely that the NADase serves to raise Ca2+ levels in the ova at appropriate times. A 1.2-kb cDNA clone containing the complete coding sequence of the native NADase protein was isolated from an unamplified ovotestis cDNA library and represents the first cloning of an NADase that generates cADPR. In vitro translation studies indicate that the protein initially has a signal sequence that may help to target it to discrete vesicles of the ova in which it is found. There are 12 cysteines in the open reading frame, two of these being in the signal sequence. No part of the sequence has significant similarity to other proteins or known nucleotide binding site consensus sequences. Northern blot analysis of poly(A)+ selected ovotestis RNA has identified an NADase mRNA of 1.85 kb. In situ hybridization analysis of cryostat sections from ovotestis has shown that the NADase mRNA is restricted to the immature ova, although the NADase protein is present in both immature and mature eggs.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.