CDAP活化多糖制备1型肺炎链球菌荚膜多糖-蛋白结合物原液的稳定性评价

目的 评价以1-氰基-4-二甲氨基-吡啶四氟硼酸盐(1-cyano-4-dimethylamino pyridinium tetrafluoroborate,CDAP)活化多糖制备的1型肺炎链球菌荚膜多糖-破伤风类毒素结合物原液的稳定性.方法 以CDAP作为活化剂,连续制备3批多糖-蛋白结合物原液,分别放置于(37±2)℃和2～8℃,定期取样检测,并评价其稳定性.结果 多糖-蛋白结合物原液在(37±2)℃条件下保存21 d,2～8℃条件下保存12个月,其主要检测指标均符合质量要求,游离多糖和游离蛋白含量分别低于30.0％和5.0％,分配系数≤0.35的多糖回收率均＞65％.结论 确定了不同条件下1型肺炎链球菌荚膜多糖-破伤风类毒素结合物原液的有效保存时间.     

Involvement of protein kinase C and tyrosine kinase in lipopolysaccharide-induced anorexia.

Injections of lipopolysaccharide (LPS, 3 microg) into the lateral ventricle elicited anorexia with fever and also decreased body weight in rats. The LPS-induced anorexia was inhibited by intracerebroventicular (i.c.v.) injections of anti-interleukin (IL)-1beta antibody (Ab), chelerythrine, genistein and tyrphostin 46, but not by injections of indomethacin. Consecutive injections of orthovanadate and LPS (0.3 microg, a dose of LPS that did not show any effect on food intake, body weight or body temperature) reduced body weight, but did not induce anorexia. On the other hand, injections of IL-1beta (50 ng) did not influence food intake, although they decreased body weight and produced fever. The IL-1beta-induced decrease in body weight was inhibited by injections of genistein, but not by injections of chelerythrine or indomethacin. These findings suggest that the LPS-induced anorexia is independent of hyperthermia and involves IL-1beta generation, tyrosine kinase (TK) and protein kinase C (PKC). This is the first in vivo evidence that activation of TK and PKC induced by LPS is linked to anorexia.     

19A型肺炎链球菌多糖结合物中游离多糖含量测定方法的建立

目的 建立19A型肺炎链球菌多糖(Streptococcus pneumoniae type 19A polysaccharide,19APS)结合物中游离PS含量的测定方法.方法 在一定条件下分别用1、3和5 g/L脱氧胆酸钠(sodium deoxycholate,DOC)溶液(pH7.3)沉淀不同浓度的破伤风类毒素(tetanus toxoid,TT),确定不同浓度DOC沉淀载体蛋白TT的浓度范围.以1 g/L DOC分离结合19 APS(19APS-TT)和游离19APS,测定上清液中的游离PS含量,并验证该DOC沉淀法的准确度和精密度.结果 分别以1和3 g/L DOC沉淀19APS-TT时,TT浓度最好分别控制在＜150和＜200μg/ml.该DOC沉淀法的准确度和精密度均良好,加标PS回收率为94.0％～107.2％,相对标准偏差均＜4％.结论 成功建立了19APS-TT中游离PS含量的测定方法.     

Roles of atypical protein kinase C in lysophosphatidic acid-induced type II adenylyl cyclase activation in RAW 264.7 macrophages

1 Lysophosphatidic acid (LPA) has been widely studied as a naturally occurring and multifunctional phospholipid messenger in diverse tissue and cell types and shown to inhibit adenylyl cyclase (AC) by a G protein-mediated mechanism. 2 In type II AC-expressing mouse RAW 264.7 macrophages, we showed that LPA at 3-50 microM increased cyclic AMP formation in a concentration-dependent manner, the effect being additive with that of forskolin or cholera toxin, and synergistic with that of prostaglandin E1 (PGE1) or isoproterenol. 3 The potentiation effect of LPA was unaffected by the removal of serum or pertussis toxin treatment. 4 Both colchicine and cytochalasin B potentiated the cyclic AMP response to PGE1, the effect being additive to that of LPA. 5 On studying the regulation of type II AC by protein kinase C (PKC), phorbol 12-myristate-13 acetate (PMA) potentiated the PGE1-elicited cyclic AMP response, this effect being non-additive to that of LPA, suggesting that PKC activation was the common mechanism involved in AC potentiation by LPA and PMA. 6 PKC inhibitor Ro 31-8220, but not Go 6976, significantly inhibited the LPA-induced cyclic AMP potentiation. 7 The potentiation effect of LPA was unaffected by long-term treatment with PMA, which resulted in the down-regulation of PKCalpha, betaI, betaII and PKCdelta, but not PKCepsilon, mu, lambda and zeta. 8 By in situ kinase assay, we found a marked increase in atypical PKC activity after LPA treatment. 9 Taken together, we conclude that LPA can elicit a unique signalling cascade in RAW 264.7 macrophages and increase type II AC activity via the activation of atypical PKC.     

Acidosis inhibits gallbladder contraction mediated by protein kinase C activation.

The effects of extracellular acidosis on gallbladder contraction were investigated using gallbladder strips isolated from guinea pigs. In an acidic medium (pH 6.9), gallbladder contraction induced by histamine and prostaglandin E2 was significantly lower than that in a normal medium (pH 7.4). Acidosis affected neither gallbladder contraction induced by histamine in the absence of extracellular Ca2+ nor that induced by KCl. Acidosis significantly inhibited Ca2+-induced contraction in the presence of sodium fluoride and phorbol 12,13-dibutyrate but not that in the presence of KCl. Staurosporine (30 nM) significantly inhibited gallbladder contraction induced by histamine and prostaglandin E2, but not that by KCl. Histamine-induced contraction in the presence of staurosporine was not affected by acidosis. Acidosis significantly inhibited Ca2+-induced contraction in the presence of histamine but not that in the presence of both histamine and staurosporine. These results suggest that extracellular acidosis selectively inhibits gallbladder contraction mediated by protein kinase C activation.     

Modulation of pulmonary macrophage superoxide release and tumoricidal activity following activation by biological response modifiers

Following immunologic activation, pulmonary macrophages may prevent or cause regression of lung metastases by mechanisms which remain largely unknown. The studies described here were designed to determine if enhanced oxygen metabolite release was related to postactivation tumoricidal activity. We have shown that in vitro activation of Fischer 344 rat pulmonary macrophages by either free or liposome-encapsulated muramyl dipeptide leads to both enhanced release of superoxide anions and marked tumoricidal activity against syngenic (Fischer 13762), allogeneic (Schmidt-Ruppin RR 1022) and xenogeneic (Fibrosarcoma MCA-F) 125I-deoxyuridine-labeled target cells. This immune modulator did not, however, metabolically activate pulmonary macrophages as effectively as liposome-encapsulated lipopolysaccharide. A 24-h in vitro incubation with either 150 U or 300 U of interferon-gamma (3 X 10(6) U/mg) or 30 U, 150 U or 300 U of interferon-alpha (6 X 10(5) U/mg) caused a significant elevation in superoxide release above controls, whereas short-term exposure (2 or 4 h) had little or no effect. Free or encapsulated 6-O-stearoyl muramyl dipeptide, on the other hand, did increase superoxide levels at all 3 time periods. When either interferon-gamma or free or encapsulated muramyl dipeptide derivative were administered to intact rats by either i.v. injection, intratracheal instillation or osmotic minipump infusion, pulmonary macrophage tumoricidal activity was observed 96 h after cell harvesting. Zymosan-stimulated superoxide release, however, was not consistently elevated above control or empty liposome treatment following this course of in vivo activation. The data collectively suggest that in vivo pulmonary macrophage activation to a tumoricidal state and metabolic activation resulting in enhanced superoxide may be separable events.     

Involvement of the protein kinase C system in calcium-force relationships in ferret aorta.

1. Intracellular Ca2+-force relationships were investigated in ferret aortic smooth muscle by the simultaneous measurement of aequorin luminescence and isometric force. Complete calcium-force curves were constructed by plotting calibrated aequorin luminescence versus force, while intracellular [Ca2+] was made to change by increasing degrees of K+ depolarization or decreasing extracellular [Ca2+]. 2. The steady state calcium-force curve in response to K+ depolarization exhibited maximal force generation at an intracellular [Ca2+] of approximately 4 x 10(-7) M. Further increases in intracellular [Ca2+] did not yield additional increments in force. 3. Protein kinase C activation with the phorbol ester, 12-deoxyphorbol-13-isobutyrate 20 acetate (DPBA) produced contractions accompanied by no detectable increases in aequorin luminescence. DPBA significantly shifted the control [Ca2+]-force relationship leftward to lower intracellular [Ca2+] with an increase in the magnitude of maximal generated force. 4. In aorta maximally precontracted by K+ depolarization, the addition of DPBA resulted in a significant increase in force in the absence of further increases in intracellular [Ca2+]. Conversely, in muscles maximally precontracted with DPBA, responses to K+ depolarization resulted in subsequent increases in force in the presence of simultaneous sustained increases in intracellular [Ca2+]. 5. The relatively specific protein kinase C antagonist H-7 caused a significant decrease in intrinsic myogenic tone in the absence of any statistically significant decrease in intracellular [Ca2+]. 6. These results suggest that protein kinase C may be an important regulator of vascular smooth muscle contractility by: (1) providing a mechanism by which the apparent [Ca2+] sensitivity of the contractile apparatus during agonist-induced contractions is increased, and (2) maintaining intrinsic myogenic tone by a mechanism the [Ca2+] requirement of which is satisfied by the resting [Ca2+]i.     

Involvement of protein kinase C and Na+/K+-ATPase in the contractile response induced by myricetin in rat isolated aorta

The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.NA:noradrenalineNA+/K+-ATPase pump:sodium-potassium-activated ATPasePKC:protein kinase CPMA:phorbol 12-myristate 13-acetateTXA2:thromboxane A2The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.     

Involvement of kappa opioid receptors in the formalin‐induced inhibition of analgesic tolerance to morphine via suppression of conventional protein kinase C activation

Objectives Repeated morphine treatment results in a decreased analgesic effect or the development of analgesic tolerance. However, we reported that some inflammatory chronic pain may inhibit morphine tolerance via kappa opioid receptor (KOR) activation. In this study, we further investigated the role of KOR in the inhibition of morphine tolerance in a chronic pain condition with a focus on the regulation of protein kinase C (PKC) activity. Methods Chronic pain was induced by formalin treatment into the dorsal part of the left hind paws of mice. The analgesic effect of morphine was measured by the tail flick method. We analysed the protein expression of PKC and its activity, and G‐protein activity of mu opioid receptor (MOR) under repeated morphine treatment with or without formalin treatment. Key findings We found that conventional subtypes of PKC (cPKC) were up‐regulated by repeated morphine treatment. Also, antisense oligonucleotide (AS‐ODN) targeting cPKC completely suppressed the development of morphine tolerance. The disappearance of the repeated morphine‐induced up‐regulation of cPKC was completely reversed by treatment with AS‐ODN targeting KOR. In addition, AS‐ODN targeting KOR significantly reversed the chronic pain‐induced down‐regulation of PKC activity or up‐regulation of MOR [³⁵S]GTPγS binding activity after repeated morphine treatment. Conclusions These results indicate that KOR plays an important role in the inhibition of repeated morphine‐induced cPKC up‐regulation under chronic pain condition. Furthermore, this may result in the increase of MOR activity and in the inhibition of morphine tolerance under chronic pain condition.     

Involvement of calcium/calmodulin‐dependent protein kinase II in methamphetamine‐induced neural damage

Methamphetamine (METH), an illicit drug, is widely abused in many parts of the world. Mounting evidence shows that METH exposure contributes to neurotoxicity, particularly for the monoaminergic neurons. However, to date, only a few studies have tried to unravel the mechanisms involved in METH‐induced non‐monoaminergic neural damage. Therefore, in the present study, we tried to explore the mechanisms for METH‐induced neural damage in cortical neurons. Our results showed that METH significantly increased intracellular [Ca²⁺]_i in Ca²⁺‐containing solution rather than Ca²⁺‐free solution. Moreover, METH also upregulated calmodulin (CaM) expression and activated CaM‐dependent protein kinase II (CaMKII). Significantly, METH‐induced neural damage can be partially retarded by CaM antagonist W7 as well as CaMKII blocker KN93. In addition, L‐type Ca²⁺ channel was also proved to be involved in METH‐induced cell damage, as nifedipine, the L‐type Ca²⁺ channel‐specific inhibitor, markedly attenuated METH‐induced neural damage. Collectively, our results suggest that Ca²⁺‐CaM‐CaMKII is involved in METH‐mediated neurotoxicity, and it might suggest a potential target for the development of therapeutic strategies for METH abuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of dietary triacylglycerol oil and diacylglycerol oil in protein kinase C activation.

The objective of the present study was to compare the effects of dietary diacylglycerol (DAG) oil with triacylglycerol (TAG) oil with a similar fatty acid composition (fatty acid chain range: C14-C22, C18 fatty acid chain: >90%) on protein kinase C (PKC) activation and on 1,2-DAG levels. Using male Wistar rats, no differences in cytosolic and membrane PKC activities in the lingual, esophageal, gastric, small intestinal, cecal, proximal colonic, and distal colonic mucosa were found between the 5% DAG and TAG oil groups, or between the 23% DAG and TAG oil groups after 1 month of feeding. The 1,2-DAG levels in the cecum and colon contents and in the feces and serum in male Wistar rats after a diet containing either 10% DAG or TAG oil feeding were similar between the groups. Moreover, exposure of Caco-2 cells to DAG and TAG oils had no effect on PKC activity in the membrane fraction, but 1,2-dioctanoyl glycerol composed of short-chain fatty acids (C8) did, suggesting the absence of an influence on PKC activity in DAG and TAG oils composed of long-chain fatty acids. In summary, the effects of DAG oil ingestion on PKC activity in the digestive tract and lingual mucosa, and on 1,2-DAG levels in the cecum and colon contents and in the feces and serum were similar to those observed for TAG oil ingestion.     

大蒜素通过激活蛋白激酶C模拟心肌缺血预适应(英文)

目的:研究大蒜素是否具有药物性预处理的作用,并探讨蛋白激酶C在大蒜素预处理中的重要作用。方法:34只离体家兔心脏挂上Langendorff装置,以95％O_2/5％CO_2混合的Krebs-Henseleit液灌流,均接受30min局部缺血和2h复灌。结果:在30min局部缺血前10min予5min药物性预处理的大蒜素组较之对照组明显缩小心肌梗塞范围(7％±6％ vs25％±7％,P<0.05),大蒜素 多粘菌素B组的心肌梗塞范围与对照组相比无显著性差异(23％±5％ vs 25％±7％,P>0.05)。结论:大蒜素具有模拟缺血预处理样保护作用,其作用可被抑制剂多粘菌素B所阻断,表明大蒜素通过激活蛋白激酶C发挥药物性预处理作用。     

Involvement of protein kinase C in the delayed cytoprotection following sublethal ischaemia in rabbit myocardium.

Rabbit hearts were preconditioned with four 5 min coronary artery occlusions 24 h before 30 min coronary occlusion with 120 min reperfusion. Preconditioning significantly reduced the percentage of myocardium infarcting within the risk zone from 49.1 +/- 4.3% to 31.8 +/- 3.5% (P < 0.05). When the protein kinase C (PKC) inhibitor, chelerythrine, was administered just before preconditioning, the delayed protection against infarction 24 h later was abolished. We conclude that the delayed cytoprotective response associated with ischaemic preconditioning of myocardium is likely to involve the early activation of one or more PKC subtypes.     

Modulation of protein kinase C by curcumin; inhibition and activation switched by calcium ions

BACKGROUND AND PURPOSE: Previous studies have identified the natural polyphenol curcumin as a protein kinase C (PKC) inhibitor. In contrast, we found significant stimulation of PKC activity following curcumin treatment. Thus, the mechanism of curcumin interaction with PKC was investigated. EXPERIMENTAL APPROACH: We employed phosphorylation assays in the presence of soluble or membrane-bound PKC substrates, followed by SDS-PAGE, autoradiography and phosphorylation intensity measurements. KEY RESULTS: Curcumin inhibited PKC in the absence of membranes whereas stimulation was observed in the presence of membranes. Further analysis indicated that curcumin decreased PKC activity by competition with Ca(2+) stimulation of the kinase, resulting in inhibition of activity at lower Ca(2+) concentrations and stimulation at higher Ca(2+) concentrations. The role of the membrane is likely to be facilitation of Ca(2+)-binding to the kinase, thus relieving the curcumin inhibition observed at limited Ca(2+) concentrations. Curcumin was found to mildly stimulate the catalytic subunit of PKC, which does not require Ca(2+) for activation. In addition, studies on Ca(2+)-independent PKC isoforms as well as another curcumin target (the sarcoplasmic reticulum Ca(2+)-ATPase) confirmed a correlation between Ca(2+) concentration and the curcumin effects. CONCLUSIONS AND IMPLICATIONS: Curcumin competes with Ca(2+) for the regulatory domain of PKC, resulting in a Ca(2+)-dependent dual effect on the kinase. We propose that curcumin interacts with the Ca(2+)-binding domains in target proteins. To our knowledge, this is the first study that defines an interaction domain for curcumin, and provides a rationale for the broad specificity of this polyphenol as a chemopreventive drug.     

Stimulation of miniature end-plate potential frequency by fluoride may involve activation of protein kinase C.

NaF caused a dose-dependent rise in miniature end-plate potential (MEPP) frequency at the frog neuromuscular junction. The effects on MEPP frequency of both NaF and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were rapidly reversed by the protein kinase C (PKC) inhibitor polymyxin B (2 microM). Theophylline augmented the response of MEPP frequency to TPA. It is concluded that the effect of fluoride on MEPP frequency may be through activation of phospholipase C and consequent PKC stimulation, and that the synergistic interaction of NaF and theophylline is consistent with such a mode of action.     

溶血性磷脂酰胆碱诱导血小板活化及蛋白质酪氨酸激酶和蛋白激酶C的作用

应用双道血小板聚集仪和荧光分光光度计分别测定溶血性磷脂酰胆碱(LysoPC)诱导的兔洗涤血小板聚集,细胞内钙离子浓度(［Ca²⁺］_i),细胞内pH值(pH_i)的变化及5-羟色胺(5-HT)的释放,并观察蛋白质酪氨酸激酶(PTK)抑制剂金雀异黄素(Gen)和蛋白激酶C(PKC)抑制剂星形孢菌素(Sta)对其作用的影响. 结果表明：LysoPC(30-300 μmol·L^-1)诱导血小板聚集,5- HT释放和［Ca²⁺］_i 升高,均呈现良好的浓度依赖性,100 μmol·L^-1以上时伴有pH_i的增加;Gen使LysoPC 诱导的聚集浓度效应曲线右移,半效聚集浓度值从(100±23) μmol·L^-1增加到(200±42) μmol·L^-1,明显抑制 ［Ca²⁺］_i 升高和胞浆碱化,对5-HT释放无明显影响;Sta对较低浓度LysoPC 诱导的血小板聚集,5-HT释放, ［Ca²⁺］_i 和pH_i的增加均有明显抑制作用,但对高浓度LysoPC 诱导的血小板活化无明显影响. 提示：LysoPC通过内Ca²⁺调节和Na^＋/H^＋交换介导血小板聚集和致密颗粒释放;PTK的激活参与了LysoPC诱导的血小板聚集,内Ca²⁺调节和Na^＋/H^＋交换,而在5-HT释放的机理中意义不大;PKC的激活在较低浓度LysoPC 诱导的血小板活化中起重要作用,高浓度LysoPC通过不依赖于PKC的途径激活血小板.     

Further evidence from an elastic artery that angiotensin II amplifies noradrenaline-induced contraction through activation of protein kinase C.

Angiotensin II (AII, 0.1 nM) increased concentration dependently the sensitivity of rabbit aortic rings to low concentrations of noradrenaline. This was not associated with increases in noradrenaline-induced 45Ca2+ uptake or efflux and was prevented by the protein kinase C (PKC) inhibitors staurosporine (0.01 microM) and calphostin C (0.1 microM). Pretreatment of the rings with PMA (phorbol-12-myristate-13-acetate, 0.1 and 1 microM, 24 h at 4 degrees C) abolished the potentiation phenomenon. We conclude that AII potentiation of noradrenaline-induced vascular tone may be due to a PKC-mediated increase in intracellular sensitivity of the contractile apparatus to Ca2+.     

Involvement of protein kinase activation in neurotrophic effects of basic fibroblast growth factor in cultured brain neurons.

The influences of K-252a and staurosporine, protein kinase inhibitors, on neurotrophic effects of basic fibroblast growth factor (bFGF) were investigated in dissociated cell cultures of the striatum, hippocampus and cerebellum of fetal rats. Addition of 1 ng/ml bFGF enhanced the survival of cultured neurons of all brain regions tested. Both K-252a (10-200 nM) and staurosporine (1-100 nM) blocked the survival promoting effects of bFGF in a concentration-dependent manner. These results suggest that bFGF exerts its neurotrophic effects through activation of protein kinase(s).     

Involvement of mitogen-activated protein kinases in Group B Streptococcus-induced macrophage apoptosis.

We previously demonstrated that Group B Streptococcus (GBS), a pathogen that causes serious neonatal infections, induces macrophage apoptosis by beta-hemolysin to avoid the host immune response. GBS-induced macrophage apoptosis is characterized by a calcium increase and is caspase-independent. This study reports the involvement of c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), three members of mitogen-activated protein kinases (MAPKs) family, in GBS-induced macrophage apoptosis. Our data indicate that during induction of apoptosis live GBS stimulates a strong persistent activation of JNK and p38 with concomitant inhibition of ERK. The time courses of MAPKs activation strongly correlate with GBS-induced macrophage apoptosis and are macrophage:GBS ratio-dependent. In fact, when GBS does not cause macrophage apoptosis, e.g. low macrophage:GBS ratio or non hemolytic GBS (gGBS), it induces a transient activation of JNK, p38, and ERK MAPKs. These latter results indicate that sustained and persistent activation of JNK and p38 and inhibition of ERK are involved in the GBS-induced macrophage apoptotic process and suggest that the time course and balance of MAPKs activation are critical for different macrophage responses to GBS (apoptosis versus antimicrobicidal activity). This study indicates a correlation between MAPKs activation and GBS-induced macrophage apoptosis. However, since neither ERK nor p38 inhibitors had an effect on GBS-induced apoptosis, their role in the complex signal network leading to GBS-induced macrophage apoptosis remains to be defined.