摘要:

AbstractMyelin basic protein, an 80‐kilodalton (kDa) protein in rat oligodendrocytes, and an 80‐kDa basic protein in neuroblastoma × neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl‐acetyl‐glycerol). TPA‐stimulated phosphorylation was inhibited by pre‐incubation with 50 μM psychosine (galactosyl‐sphingosine), confirming that it is mediated through the phospholipid‐dependent protein kinase C (PK‐C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic‐AMP‐dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte‐specific 2′,3′‐cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.:Proceedings of the National Academy of Sciences of the United States of America85:939, 1988)[1]. The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol f

ISSN:0730-2312    

DOI:10.1002/jcb.240400302

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

年代:1989

数据来源: WILEY

摘要:

AbstractBALB/c 3T3 cells infected with a temperature‐sensitive mutant (LA90) of RSV have been used to investigate possible heterologous interactions between the pp60v‐srctyrosyl kinase and the epidermal growth factor (EGF) and bradykinin receptors. The LA90 pp60v‐srcexhibits a very rapid activation t1/2(<5 min) of protein kinase activity on decreasing the temperature from 40°C to 35°C. This change in temperature was also found to induce a very rapid decrease in the affinity for125I‐EGF of receptors on the RSV‐LA90‐infected cells but not of those on control parental cells. However, no significant changes were detected in the binding of3H‐bradykinin to either cell line. Two separable processes control the desensitization of the EGF receptor by pp60v‐src, both of which are independent of protein kinase C. The first is rapid and transient, while the second is sensitive to cycloheximide and persists long after inactiva

ISSN:0730-2312    

DOI:10.1002/jcb.240400303

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe X‐ray structures of pancreatic bovine and porcine phospholipases A2 have been used along with interactive computer graphics to design conformationally rigid, novel compounds (1‐meta‐hydroxybenzyl‐2‐substituted acenaphthenes) directed at the active sites of these enzymes. In vitro testing confirmed that the designed compounds are potent inhibitors of the porcine pancreatic phospholipase A2 and exhibit both stereoselectivity and structure‐activity relationships that are consistent with the proposed mode of binding. These compounds take advantage of a hydrophobic “slot” positioned between residues Leu‐2 and Tyr‐69 while positioning hydrogen‐bonding functionality directed at the nd1‐N of His‐48. Experimental evidence shows a regioselective preference for this H‐bond acceptor. A second part of the strategy used a tethered amine to displace the essential calcium pro

ISSN:0730-2312    

DOI:10.1002/jcb.240400304

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

年代:1989

数据来源: WILEY

摘要:

AbstractModulation of neutrophil activation by catecholamines reflects a fine‐tuning by coupling inhibitory and stimulatory receptor pathways. The catecholamine isoproterenol (ISO) binds to beta‐adrenergic cell surface receptors and thereby inhibits cell responses such as O 2−production stimulated by formyl peptides. However, ISO did not inhibit O 2−generation activated by 1 μM ionophore A23187, the protein kinase C activators phorbol ester (PMA, 100 ng/ml) and oleoylacetylglycerol (OAG, 50 μM), and the G‐protein activator NaF (40 mM). Furthermore, the overall kinetics of oxidant production in the presence of ISO were unchanged when cells were stimulated with PMA, OAG, A23187, and NaF. These results would imply that neither intracellular calcium, the activation of protein kinase C, nor the activation of G‐protein are the primary target of the inhibitory pathway. Accordingly, pertussis toxin did not block PMA or NaF‐stimulated superoxide generation. In contrast, formyl peptide‐dependent GTPase activity is inhibited by ISO in sonicated cell preparations. Since ISO increases the cAMP concentration in the cell, the possibility is raised that a cAMP‐dependent kinase inhibits signal transduction in part by blocking the interaction of this rece

ISSN:0730-2312    

DOI:10.1002/jcb.240400305

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe phosphorylation of an Mr 82,000 protein (p82) in the Triton X‐100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)‐catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82‐kinase system is down‐modulated after TPA treatment of mouse skin, with a half‐life of around 5 h. Down‐modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down‐modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the

ISSN:0730-2312    

DOI:10.1002/jcb.240400306

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

年代:1989

数据来源: WILEY

摘要:

AbstractA gene coding for the (pro)phospholipase A2(PLA2) from bovine pancreas has been designed, synthesized, and expressed inEscherichia coli. The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies. Codons occurring frequently in prokaryotic systems were chosen whenever possible. The total gene spans 404 base pairs and was divided into 33 oligonucleotides. The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13‐ as the cloning vehicle. The two fragments were then ligated and cloned into pBSM13‐ to complete the gene. The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing. The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from theE. colisecretion vector pIN‐III‐ompA3. Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro‐peptide of proPLA2. The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2. The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase

ISSN:0730-2312    

DOI:10.1002/jcb.240400307

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

年代:1989

数据来源: WILEY

摘要:

AbstractInositol phosphate action in an intact cell has been investigated by intracellular microinjection of eight inositol phosphate derivatives intoXenopus laevisoocytes. These cells have calcium‐regulated chloride channels but do not have a calcium‐induced calcium release system. Microinjection of inositol 1,3,4,5‐tetrakisphosphate (IP4), inositol 1,2‐(cyclic)‐4,5‐trisphosphate (cIP3), inositol 1,4,5‐trisphosphate (IP3), or inositol 4,5‐bisphosphate [(4,5)IP2], open chloride channels to induce a membrane depolarization. However, inositol 1‐phosphate (IP1), inositol 1,3,4,5,6‐pentakisphosphate (IP5), inositol 1,4‐bisphosphate, or inositol 3,4‐bisphosphate are unable to induce this depolarization. The depolarization is mimicked by calcium microinjection, inhibited by EGTA coinjection, and is insensitive to removal of extracellular calcium. By means of the depolarization response, the efficacy of various inositol phosphate derivatives are compared. IP3and cIP3induce similar half‐maximal, biphasic depolarization responses at an intracellular concentration of approximately 90 nM, whereas IP4induces a mono‐ or biphasic depolarization at approximately 3400 nM. At concentrations similar to that required for IP3and cIP3, (4,5)IP2induces a long‐term (greater than 40 min) depolarization. The efficacy (cIP3= IP3= (4,5)IP2≫ IP4) and action of the various inositol phosphates in an intact cell and their inability to induce meio

ISSN:0730-2312    

DOI:10.1002/jcb.240400308

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

年代:1989

数据来源: WILEY

摘要:

AbstractMicrosomal membranes from carrot suspension cells were phosphorylated in vitro with [γ‐32P]ATP. In the presence of submicromolar concentrations of the natural auxin indoleacetic acid (IAA), a rapid, but transient decrease of the [32P] label could be detected in the phospholipid extracts of the membranes. The phytohormone effect was not the result of an inhibition of the lipid phosphorylation reactions, but was caused by a simultaneous release of water‐soluble compounds, which, according to their chromatographic properties, were assumed to contain inositol polyphosphates. Although the [32P]‐labeled lipids, as well as the inositol polyphosphates, were not identified unequivocally by chemical analysis, these findings point to an auxin‐mediated control of a phosphoinositidase C‐like reaction similar to the hormone‐stimulated phosphoinositide response in animals. Exogenously applied inositol (1,4,5)trisphosphate [(1,4,5)IP3] was found to release45Ca2+from preloaded membrane vesicles of carrot cells. Both the detection of the auxin‐stimulated phosphoinositide response and the (1,4,5)IP3‐mediated Ca2+release on isolated cell membranes offer new experimental approaches for the identification of the putative auxin receptor and its signal tran

ISSN:0730-2312    

DOI:10.1002/jcb.240400309

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

年代:1989

数据来源: WILEY

摘要:

AbstractA23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5‐lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187‐stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP‐stimulated neutro

ISSN:0730-2312    

DOI:10.1002/jcb.240400310

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe biosynthesis of platelet‐activating factor (PAF‐acether or 1‐O‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs‐II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl‐GPC, the source of PAF‐precursor alkyl‐lyso‐GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2in providing lyso‐PAF to the acetyltr

ISSN:0730-2312    

DOI:10.1002/jcb.240400311

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

年代:1989

数据来源: WILEY