NO对新生大鼠心肌细胞PKC活性的影响

利用培养新生大鼠心肌细胞，检测NO前体L-精氨酸(L-arginine,L-Arg)和NO供体硝普钠(sodium nitroprusside,SNP)对PKC活性的影响，并探讨内、外源性NO在PKC激动剂佛波指(phorbol 12-myristate 13-acetate,PMA)激活PKC中的作用.实验结果表明：培养基中加入L-Arg，PKC活性呈剂量依赖性降低；用L-Arg进行预处理，30 min后加入PMA，PKC活性明显降低，与单纯PMA组相比有显著差异；NOS抑制剂L-NAME本身对基础状态PKC活性无明显影响，但可阻断L-Arg对上述2个效应的影响；培养液中加入NO供体SNP，PKC活性呈剂量依赖性降低；用SNP预处理心肌细胞，5 min后加入PMA，PKC活性与单纯PMA组相比有显著性差异.以上结果表明，内、外源性NO均具有剂量依赖性抑制PKC活性的作用，PKC可能是NO对心肌细胞作用的胞内信号传导通路的关键部位或重要信号分子之一；L-Arg通过NOS先生成NO，NO再对PKC起抑制作用.     

v-Fos transformation effector binds with CD2 cytoplasmic tail

The CD2 molecule, an adhesive and co-stimulating molecule, expresses virtually on all T lymphocytes, thymocytes, as well as natural killer cells, playing an important role in thymus development, T cell activation and apoptosis[1―3]. CD2 molecule consists of an ex- tracellular region with two Ig-like domains, a single membrane-spanning domain, and a positively charged proline-rich cytoplasmic tail, which is highly conserved between different species[1,4―7]. The functions of CD2 in signal t…     

Crystal structure of staphylococcal enterotoxin B, a superantigen.

The three-dimensional structure of staphylococcal enterotoxin B, which is both a toxin and a super-antigen, has been determined to a resolution of 2.5 A. The unusual main-chain fold containing two domains may represent a general motif adopted by all staphylococcal enterotoxins. The T-cell receptor binding site encompasses a shallow cavity formed by both domains. The MHCII molecule binds to an adjacent site. Another cavity with possible biological activity was also identified.     

用流式细胞术研究PKC及VRP，CsA对多药抗性细胞的调制与逆转

利用流式细胞术检测抗药性细胞内Ｒｈｏ－１２３的荧光强度作为检测抗性细胞抗性程度的指标，研究了蛋白激酶Ｃ（ＰＫＣ）抑制剂Ｈ７，肿瘤促进剂佛波酯（ＴＰＡ）对多药抗性细胞的调节；钙离子通道阻断剂维拉帕米（ＶＲＰ），免疫抑制剂（ＣｓＡ）对多药抗性的逆转作用．测定了抗性细胞细胞膜和胞浆ＰＫＣ活性水平，指出ＰＫＣ可能通过磷酸化细胞膜上的Ｐ－糖蛋白（Ｐ－ｇｐ）来调节多药抗性细胞的抗性水平，并提供了一种筛选多药抗性逆转药物的简便方法．     

Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes

The lymphocyte function-associated molecule LFA-1 (CD11a/CD18) plays a key part in lymphocyte adhesion. Lymphocytes do not adhere spontaneously; activation of protein kinase C (PKC) by phorbol esters, however, gives rise to strong LFA-1-dependent adhesion, indicating that activation of LFA-1 is required to induce cell adhesion. We have now investigated whether the functionally important CD2 and CD3 surface structures on T lymphocytes are involved in the activation of LFA-1. The stimulation of these molecules, which causes activation of PKC, strongly promoted LFA-1-dependent adhesion. Furthermore, we demonstrate by using cells from an LFA-1-deficient patient that this enhanced lymphocyte adhesion is caused by activation of the LFA-1 molecule and not by activation of its ligands. LFA-1 was persistently activated by triggering through CD2 but only transiently by triggering through CD3. We postulate that CD2 and CD3 can differentially regulate the affinity of LFA-1 for its ligands by modulating its molecular conformation through PKC-dependent mechanisms.     

Protein kinase C mediates neural induction in Xenopus laevis

Inductive cell interactions are essential in early embryonic development, but virtually nothing is known about the molecular mechanisms involved. Recently factors resembling fibroblast growth factor and transforming growth factor-beta were shown to be involved in mesoderm induction in Xenopus laevis, suggesting that membrane receptor-mediated signal transduction is important in induction processes. Here we report direct measurements of protein kinase C (PKC) activity in uninduced ectoderm, and in neuroectoderm shortly after induction by the involuting mesoderm, in Xenopus laevis embryos. Membrane-bound PKC activity increased three to fourfold in the induced neuroectoderm while the cytosolic PKC activity was decreasing, indicating that PKC activity was translocated during neural induction. A similar time- and dose-dependent translocation of activity was seen after incubation with the PKC activator 12-O-tetradecanoyl phorbol-13-acetate, which also induced neural tissue in competent ectoderm, suggesting that PKC is involved in the response to the endogenous inducing signal during neural induction.     

Effect of nerve regeneration factor on differentiation of PC12 cells and its signaling pathway

The effects of nerve regeneration factor (NRF) on neuronal differentiation of PC12 cells and its signaling pathway are investigated by morphological observation and immunofluorescent cytochemical method, and the activity of ERK1/2 in NRF-treated PC12 cells in absence of serum is also studied by immuno-coprecipitation and Western blot analysis. The MEK1/2-specific inhibitor U0126, the broad-spectrum protein kinase C (PKC) inhibitor G?6983 and tyrosine protein kinase (TPK) inhibitor genistein were used to determine the roles of the activation of ERK1/2 by NRF and the involvement of certain kinds of PKC or TPK receptor in this activation process. The results show that U0126 and G?6983 inhibit the activation of ERK1/2 by NRF to different extents, while genistein has no effect on it, demonstrating that NRF remarkably induces neuronal differentiation of PC12 cells through activating ERK1/2 in a dose-dependent and time-dependent manner.     

多药耐药基因与肿瘤

介绍多药耐药（MDR）基因结构和表达、在肿瘤组织中的表达、检测方法及MDR逆转。指出研究MDR基因与肿瘤相互关系,除了具有理论意义外,还可为临床提高肿瘤化疗效果、正确选择治疗方案提供新思路。     

Effect of thrombin on the apoptosis of hippocampal neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activating peptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. The numbers of apoptotic cell and apoptotic rate of hippocampal neurons treated by different concentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method and Flow Cytometry. When the concentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neurons reached peak value, were 27.3±4.0 and (29.333±4.633)%, respectively. Immunocytochemistry assay show that Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effect of thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombin induced hippocampal neuron apoptosis in a dose-dependent manner through activating protease-activated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related with the effect of high concentration thrombin induced apoptosis on hippocampal neurons. Foundation item: Supported by the Natural Science Foundation of Hainan Province (N30215) Biography: YANG Wen-qiong (1968-), female, Ph.D. candidate, research direction: cerebrovascular disease.     

Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles. Kinesin, a microtubule-based motility protein which may participate in organelle transport and mitosis, binds microtubules in a nucleotide-sensitive manner, and requires hydrolysable nucleotides to translocate microtubules over a glass surface. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5'-adenylyl imidodiphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg2+ with EDTA, by addition of Na3VO4, or by addition of AMPPNP with or without Mg2+. Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg2+-ATPase activity.     

Potentiation of synaptic transmission in the hippocampus by phorbol esters

Protein kinase C (PKC), a calcium-dependent phospholipid-sensitive kinase which is selectively activated by phorbol esters, is thought to play an important role in several cellular processes. In mammalian brain PKC is present in high concentrations and has been shown to phosphorylate several substrate phosphoproteins, one of which may be involved in the generation of long-term potentiation (LTP), a long-lasting increase in synaptic efficacy evoked by brief, high-frequency stimulation. Since the hippocampus contains one of the brain's highest levels of binding sites for phorbol esters and is the site where LTP has been most thoroughly characterized, we examined the effects of phorbol esters on hippocampal synaptic transmission and LTP. We found that phorbol esters profoundly potentiate excitatory synaptic transmission in the hippocampus in a manner that appears indistinguishable from LTP. Furthermore, after maximal synaptic enhancement by phorbol esters, LTP can no longer be elicited. Although the site of synaptic enhancement during LTP is not clearly established, phorbol esters appear to potentiate synaptic transmission by acting primarily at a presynaptic locus since changes in the postsynaptic responses to the putative transmitter, glutamate, cannot account for the increased synaptic responses induced by phorbol esters. These findings, in conjunction with previous biochemical studies, raise the possibility that, in mammalian brain, PKC plays a role in controlling the release of neurotransmitter and may be involved in the generation of LTP.     

MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin.

AGONISTS that stimulate protein kinase C (PKC) induce profound changes in cell morphology correlating with the reorganization of submembranous actin, but no direct connection between PKC and actin assembly has been identified. The myristoylated, alanine-rich C kinase substrate (MARCKS) binds calmodulin and is a predominant, specific substrate of PKC which is phosphorylated during macrophage and neutrophil activation , growth factor-dependent mitogenesis and neurosecretion; it is redistributed from plasma membrane to cytoplasm when phosphorylated and is involved in leukocyte motility. Here we report that MARCKS is a filamentous (F) actin crosslinking protein, with activity that is inhibited by PKC-mediated phosphorylation and by binding to calcium-calmodulin. MARCKS may be a regulated crossbridge between actin and the plasma membrane, and modulation of the actin crosslinking activity of the MARCKS protein by calmodulin and phosphorylation represents a potential convergence of the calcium-calmodulin and PKC signal transduction pathways in the regulation of the actin cytoskeleton.     

ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats

Transposons are discrete segments of DNA which are capable of moving from one site in a genome to many different sites. Tn3 is a prokaryotic transposon which is 4,957 base pairs (bp) long and encodes a transposase protein which is essential for transposition. We report here a simple method for purifying Tn3 transposase and demonstrate that the transposase protein binds specifically to the ends of the Tn3 transposon in an ATP-dependent manner. The transposase protein binds to linear double-stranded DNA both nonspecifically and specifically; the nonspecific DNA binding activity is sensitive to challenge with heparin. Site-specific DNA binding to the ends (inverted repeats) of Tn3 is observed only when binding is performed in the presence of ATP; this ATP-dependent site-specific DNA binding activity is resistant to heparin challenge. Our results indicate that ATP qualitatively alters the DNA binding activity of the transposase protein so that the protein is able to bind specifically to the ends of the Tn3 transposon.     

一种基于Propidium Monoazide与PCR结合的选择性检测细菌活细胞的方法

建立了一种PMA与PCR结合的细菌活细胞检测的方法,可有效抑制微生物死细胞DNA的PCR扩增,从而排除死细胞对微生物活细胞检测的影响．结果表明,PMA浓度大于3 μg/ml时可抑制死细胞DNA的PCR扩增,PMA浓度高达50 μg/ml时仍不影响活细胞DNA的PCR扩增;并且得出曝光时间大于3 min可使PMA与死细胞DNA分子共价交联,抑制其PCR扩增;浊度影响PMA交联的结果表明浊度小于10 NTU时,PMA仍能有效地抑制死细胞DNA的PCR扩增,而当浊度大于100 NTU时,PMA失去效果．     

Alpha-lactalbumin possesses a novel calcium binding loop

Calcium performs a unique role in biology, achieving biological effects through highly specific interactions with and modulation of target proteins. It has been proposed that calcium-modulated proteins possess a characteristic, evolutionarily related, binding fold, known as the EF-hand. The high-resolution X-ray structure of alpha-lactalbumin reveals a Ca2+ binding fold that resembles an EF-hand only superficially and presumably has no evolutionary relationship with it. However, there is clear homology with the corresponding loop in c-type lysozyme (the 'parent' molecule of alpha-lactalbumin). This study, at 1.7 A resolution, represents one of the most accurate analyses of a calcium binding protein yet reported.     

Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly

The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.     

Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.     

Using of the surface plasmon resonance cytosensor for real-time and non-invasive monitoring of cellular effects in living C6 cells induced by PMA

By cell and molecular biology, the traditional life science research has been improved into modern ex-perimental science. Because cell is the basic structural and functional unit of the mysterious life, the study of life phenomena will be ultimately embodied into the study of cell biology. How to acquire a dynamic knowledge of the intricate biomolecular reactivity and process in living cells has become one of the hotspots in recent biological researches. For the complexity of cellular effects,…     

Induction of vanilloid receptor channel activity by protein kinase C

Capsaicin or vanilloid receptors (VRs) participate in the sensation of thermal and inflammatory pain. The cloned (VR1) and native VRs are non-selective cation channels directly activated by harmful heat, extracellular protons and vanilloid compounds. However, considerable attention has been focused on identifying other signalling pathways in VR activation; it is known that VR1 is also expressed in non-sensory tissue and may mediate inflammatory rather than acute thermal pain. Here we show that activation of protein kinase C (PKC) induces VR1 channel activity at room temperature in the absence of any other agonist. We also observed this effect in native VRs from sensory neurons, and phorbol esters induced a vanilloid-sensitive Ca2+ rise in these cells. Moreover, the pro-inflammatory peptide, bradykinin, and the putative endogenous ligand, anandamide, respectively induced and enhanced VR activity, in a PKC-dependent manner. These results suggest that PKC may link a range of stimuli to the activation of VRs.