Adaptation of wild-type measles virus to cotton rat lung cells: E89K mutation in matrix protein contributes to its fitness

Wild-type measles virus (wtMeV) adapted well to cotton rat lung (CRL) cells after serial passages. In order to evaluate the contributions of the individual genes of wtMeV for adaptation, whole genome sequences of the adapted and original viruses were determined and analyzed. The results showed that there were two mutations in the whole genome of the adapted virus. One mutation was located at the 265th nucleotide in the open reading frame (ORF) of the M gene, resulting in the substitution of the 89th amino acid from E (glutamate) to K (lysine). The other was a silent mutation located at the 4182nd nucleotide in the ORF of the L gene. It was demonstrated that the E89K mutation in the M protein is responsible for the adaptation of wtMeV MV99Y in CRL cells. Cotton rats were infected with adapted virus and the original strain via intranasal inoculation. Virus titer results showed that adapted strain replicated better than the original strain in cotton rat lungs. It is suggested that the E89K mutation also contributes to the enhancement of wtMeV replication in a cotton rat model infected intranasally. The results revealed that the E89K mutation in the M protein plays a key role in wtMeV adaptation in cotton rat and CRL cells.     

Translocation of regucalcin to rat liver nucleus: involvement of nuclear protein kinase and protein phosphatase regulation

The translocation of regucalcin to the nuclei of normal rat liver was investigated. The existence of endogenous regucalcin in isolated liver nuclei was confirmed by Western blotting using anti-regucalcin antibody. Nuclear translocation of regucalcin was estimated by sodium sulfate-polyacrylamide gel electrophoresis analysis. When isolated liver nuclei were incubated in the presence of exogenous regucalcin (50 microg/ml; 1.5 microM), potent band for regucalcin was found in the nuclei, indicating that the protein is translocated into the nucleus. This translocation was an early event. Nuclear regucalcin translocation was not appreciably changed in the presence of adenosine 5'-triphosphate (2 mM), guanosine 5'-triphosphate (2 mM), calcium chloride (0.1 mM), and the lectin wheat germ agglutinin (50 or 100 microg/ml), suggesting that its translocation is not mediated through nuclear localization signal. Moreover, Ca2+-dependent protein kinase and protein tyrosine phosphatase activities in isolated liver nuclei were significantly increased in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture, and these increases were completely abolished by the addition of regucalcin (50 microg/ml). This study demonstrates that regucalcin is translocated into liver nucleus, and that it can regulate the nuclear function.     

Ⅳ.Nogo作为一种浆膜蛋白发挥其轴突再生抑制作用

哺乳动物的外周神经系统轴突损伤后可以再生,但在中枢神经系统却不同.中枢一些种类的神经突可以在外周神经移植物中延伸相当长的距离[1].通过对中枢和外周的髓鞘对比可以发现中枢神经白质蛋白选择性地抑制轴突的生长[2].     

Effects of a protein phosphatase inhibitor,okadaic acid,on membrane currents of isolated guinea-pig cardiac myocytes

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca²⁺ current (I _Ca) was recorded as peak inward current in response to test pulse to O mV. Extracellular application of OA (5–100M) produced an increase ofI _Ca. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline.I _Ca was increased from 11.3±0.8A cm^–2 to 19.0±1.1A cm^–2 (n=4) by 5M-OA in the presence of 1nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the -adrenergic increase ofI _Ca was markedly prolonged by OA. The -adrenergic stimulation of cardiac Ca²⁺ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA onI _Ca is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.     

Bifunctional role of the leishmanial antimonate reductase LmACR2 as a protein tyrosine phosphatase

LmACR2 is the first identified antimonate reductase responsible for the reduction of pentavalent antimony in pentostam to the active trivalent form of the drug in Leishmania. LmACR2 is a homologue of the yeast arsenate reductase Acr2p and Cdc25 phosphatases and has the HC[X]5R phosphatase motif. Purified LmACR2 exhibited phosphatase activity in vitro and was able to dephosphorylate a phosphotyrosine residue from a synthetic peptide. This phosphatase activity was inhibited by classical inhibitors such as orthovanadate. LmACR2-catalyzed phosphatase activity was inhibited by either antimonate or arsenate. Site-directed mutagenesis experiments showed that the H74C[X]5R81 motif was involved in catalysis. This is the first report of a metalloid reductase with a bifunctional role in protein tyrosine phosphatase activity. Leishmania is never exposed to metalloids during its life cycle. It is therefore unlikely that it would evolve an enzyme exclusively for drug activation. We propose that the physiological function of LmACR2 is to dephosphorylate phosphotyrosine residues in leishmanial proteins.     

Mouse brain localization of the protein kinase C-enhanced phosphatase 1 inhibitor KEPI (kinase C-enhanced PP1 inhibitor)

We recently identified the protein kinase C-enhanced protein phosphatase 1 (PP1) inhibitor KEPI based on its morphine-induced upregulation in striatum. Regulation of protein serine/threonine dephosphorylation by PP1 can modulate important brain signaling pathways. To improve understanding of KEPI's role in the brain, we have developed anti-KEPI sera in rabbits immunized with a hemocyanin conjugate of KEPI residues 66-80, characterized the specificity that this serum provides, mapped the distribution of immunoreactive KEPI (iKEPI) in mouse brain, rat dorsal root ganglia and striatal cultures and documented KEPI binding to PP1 in vitro. Staining is found in apparently neuronal processes and, often less intensely, in neuronal perikarya in primary cultures and in neurons and neuronal elements from a number of brain regions. iKEPI fiber/terminal patterns are relatively densely distributed in striatum, nucleus accumbens, septum, bed nucleus of the stria terminalis, hippocampus, paraventricular thalamus, ventromedial hypothalamus, interpeduncular nucleus, raphe nuclei, nucleus caudalis of the spinal tract of the trigeminal and dorsal horn of the spinal cord. iKEPI-positive cell bodies lie in the nucleus accumbens, striatum, lateral septal nucleus, granular layer of dentate gyrus, interpeduncular nucleus, dorsal root ganglia and cerebellar vermis. These expression patterns point to possible roles for KEPI in regulating protein dephosphorylation by inhibiting PP1 activities in a number of brain pathways likely to use several different neurotransmitters and to participate in a number of brain functions. Dense KEPI immunoreactivity in nucleus accumbens perikarya, combined with evidence for its regulation by opiates, supports possible roles for KEPI in molecular signal transduction pathways important for drug reward and addiction.     

Okadaic acid, a protein phosphatase inhibitor, increases the calcium transients in isolated ferret ventricular muscle.

Okadaic acid is a protein phosphatase inhibitor which has been found to produce a marked positive inotropic effect in isolated cardiac muscle. Using aequorin-injected ferret papillary muscles, we demonstrate that the increase in tension seen with okadaic acid is accompanied by a simultaneous increase in the amplitude of the calcium transients. By comparison with the effects of changing the extracellular calcium concentration, it is shown that the increase in calcium transient amplitude can account for the inotropic effect of okadaic acid.     

The expression and localization of a novel protein phosphatase inhibitor 2810408A11Rik in mouse testis and sperm

This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm,and explored its role in spermatogenesis and sperm function.The expression levels of 2810408A11Rik mRNA in multiple tissue samples were analyzed using bioinformatic resources and RT-PCR technique.A specific rabbit polyclonal antibody was prepared by prokaryotic expression of 2810408A11Rik recombinant protein and utilized for animal immunization.Western blotting,immunohistochemistry and immunofluorescence were used to detect the expression and distribution of 2810408A11Rik.The results of the bioinformatic analysis and RT-PCR showed that 2810408A11Rik mRNA was specifically expressed in mouse testis,and 2810408A11Rik protein included a protein phosphatase inhibitor domain.Western blotting assays,immunohistochemistry and immunofluorescence confirmed the expression of 2810408A11Rik protein in mouse testis,especially in post-meiosis round and long spermatids,and that it is localized in the acrosome and the post-nucleus area of sperm.Our findings suggest that 2810408A11Rik may play an important role in spermatogenesis,sperm capacitation and fertilization.     

Attempts to isolate a kallikrein inhibitor from rat salivary gland

Besides the oxytocic principle (kallikrein) it was possible to separate from the rat submandibular gland extract an inhibitor presenting a specific antagonism against the oxytocic action of the submandibular gland kallikrein.A protein fraction collected from sephadex G-100 column showed maximal oxytocic activity, with an OD at about 500 and an inhibitory effect with an OD at about 1700, indicated by absorption at 280 m.Since the inhibitor appears in fractions following those holding the kallikrein activity, it is suggested that it has a lower molecular size than kallikrein.The concentration of the inhibitor in the gland increases parallel with the development of animal.     

Functions of human complement inhibitor C4b-binding protein in relation to its structure

Considering the destructive potential of the complement cascade, it is no surprise that there are several complement inhibitors present in blood and expressed on virtually all cells of the body to protect self tissue. C4b-binding protein (C4BP) is a potent soluble inhibitor of the classical and lectin pathways of complement. This large (500 kDa) plasma glycoprotein consists of seven identical 75 kDa alpha-chains and a unique 40 kDa beta-chain that are held together by disulphide bridges. Both types of subunit are almost exclusively composed of complement control protein (CCP) domains. In recent years, detailed studies of structure-function relationships have yielded new understanding of the interactions between C4BP and the activated complement factors C4b and C3b, heparin, and vitamin K-dependent anticoagulant protein S. This review describes the localization of binding sites for a number of C4BP ligands in relation to well-established and novel functions of C4BP such as complement inhibition, protection of apoptotic cells from complement, CD40-dependent stimulation of B cells, and the contribution of a number of human pathogens to pathogenesis.     

Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1(IIIB) entry and replication with EC(50) values of 3.92+/-0.62 and 6.59+/-1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1(KM018) with EC(50) values of 44.44+/-10.20 nM, as well as suppressing HIV-1-induced cytopathic effect with an EC(50) value of 3.04+/-1.20 nM. It also inhibited HIV-2(ROD) and HIV-2(CBL-20) entry and replication in the microM range. Notably, HR212 was highly effective against T20-resistant strains with EC(50) values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/AIDS patients, particularly for those infected by T20-resistant variants.     

Swelling as a possible mechanism of action of X537A to release histamine from rat mast cells

Conclusions The present results strengthen the view that the mechanism of histamine release induced by X537A is different from that induced by agents which depend on calcium and metabolic energy for their action. The swelling of the cells seems to be inherent in the secretory action of X537A on rat mast cells. The fact that X537A is a carrier of monovalent cations [4] can well explain its swelling action and might be the ultimate reason for release. The possibility of a more active role of X537A as an aminophore [4] must be taken into account and is under further investigation.     

冠状动脉粥样硬化性心脏病患者凝血酶激活的纤溶抑制物及其编码区基因多态性的研究

目的 探讨凝血酶激活的纤溶抑制物(thrombin aetiralable fibrinolysis inhibitor,TAFI)及其编码基因CPB2单核苷酸多态性与冠状动脉粥样硬化性心脏病(冠心病)之间的联系.方法 应用聚合酶链反应-限制性片段长度多态性分析技术(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)检测210例冠心病患者和190名正常对照者的TAFI基因编码区Thr325Ile、Tnr147Ala多态性分布特点,同时应用发色底物法和ELISA法分别测定TAFI的活性及抗原,并进一步分析基因多态性与TAFI的活性及抗原之间的关系.结果 冠心病组(心肌梗死组及心绞痛组)血浆中TAFI的活性及抗原水平均较对照组显著增高,差异有统计学意义.CPB2基因C1040T(Thr325Ile)及G505A(Thr147Ala)2个位点的3种基因型在冠心病组和对照组的频率分布分别为C1040C(Thr325Thr)67(31.9%)、64(33.6%);C1040T(Thr325Ile)109(51.9%)、92(48.4%);T1040T(Ile325Ile)34(16.2%)、34(17.8%);G505G(Ala147 Ala)75(35.7%)、72(37.8%);G505A(Thr147Ala)112(53.3%)、96(50.5%);A505A(Thr147Thr)23(10.9%)、22(11.7%),经x2检验,基因型分布符合Hardy-Weinberg平衡,并且两组之间各种基因型频率分布差异无统计学意义(P>0.05).在冠心病组和对照组Thr325Ile不同的基因型对TAFI活性没有影响;对TAFI抗原含量的影响则以Thr325Thr纯合基因型者血浆TAFI抗原浓度最高,较其他两型差异有统计学意义(P<0.05),Thr325Ile与TIle325Ile型之间则差异无统计学意义(P>0.05).而Tnr147Ala基因多态性与血浆中TAFI活性及抗原水平之间的差异均无统计学意义(P>0.05).结论 TAFI具有抑制纤溶的作用,可能是冠心病发病的危险因子.TAFI编码区基因Thr325Ile的多态性对血浆中TAFI抗原水平有明显影响,但Thr325Ile、Thr147Ala的多态性与冠心病的发生没有明显的相关性.     

大鼠脊髓iNOS的过表达参与形成带状疱疹后神经痛

目的:观察诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)在带状疱疹后神经痛(postherpetic neuralgia;PHN)大鼠脊髓的表达变化以及与痛行为的相关性。方法:SD大鼠随机分为水痘带状疱疹病毒感染组(varicella zoster virus,VZV组),假接种组(mock infected,Mock组)和空白对照组(Naive组);每组在接种前和接种后3、5、7、10、14、17、21 d先进行痛觉行为学检测,然后利用免疫组化染色和Western Blot检测脊髓iNOS以及NOS的其它两种同功酶nNOS和eNOS的表达变化;在痛觉阈值最低的时间点,鞘内注入各种NOS抑制剂进行行为药理学检测,同时在脊髓进行iNOS与NeuN,GFAP或OX-42的免疫荧光双重染色。结果:与Mock组和Naive组比较,VZV组在病毒注射后5 d形成了显著的机械性异常疼痛,14 d达到巅峰(P0.05)。VZV组脊髓背角iNOS染色密度和表达量显著增加(P0.05),免疫荧光双重染色显示iNOS由星形胶质细胞生成。iNOS的表达增加与痛觉阈值的降低显著相关(P0.001,r=-0.89)。VZV组脊髓内nNOS和eNOS的表达没有变化。VZV组鞘内给予iNOS抑制剂L-NIL或NO清除剂PTIO能够有效镇痛,而nNOS或eNOS的抑制剂7-NINA,L-NIO则无镇痛作用。结论:脊髓背角iNOS的上调参与了PHN大鼠痛敏状态的形成。     

Effect of BQ123 on vasoconstriction as a result of either hypoxia or endothelin-1 in perfused rat lungs

A possible role of endothelin (ET)-1 in mediating hypoxic pulmonary vasoconstriction (HPV) was examined by comparing haemodynamic differences between ET-1-induced vasoconstriction and HPV in isolated perfused rat lungs. An ET_A receptor antagonist (BQ123) was also employed to assess the effects of ET-1. The pulmonary arterial pressure (Ppa) was significantly increased by alveolar hypoxia (3% O₂) and by ET-1 (5 nM). The pulmonary microvascular pressure was not changed by hypoxia, but increased more than two-fold by ET-1 (P < 0.01). Hypoxia significantly increased pulmonary arterial resistance (P < 0.01) while ET-1 significantly increased pulmonary venous resistance (P < 0.01), and slightly increased arterial resistance. Lung weight was increased by ET-1 and decreased by hypoxia, accompanied by similar Ppa responses in both cases. BQ123 (10^-6 m and 10^-5 m ) did not influence the changes in Ppa and lung weight induced by hypoxia or angiotensin II (0.3 μg). BQ123 did, however, suppress (P < 0.05) the increase in Ppa and lung weight induced by 5 nM ET-1. Thus, it appears unlikely that ET-1 is involved in changes in pulmonary vascular tone during acute HPV.     

Calcineurin (protein phosphatase 2B) is involved in the mechanisms of action of antidepressants

Calcineurin (PP2B) is a Ca(2+)-dependent protein phosphatase enriched in the brain that takes part in intracellular signaling pathways regulating synaptic plasticity and neuronal functions. Calcineurin-dependent pathways are important for complex brain functions such as learning and memory. More recently, they have been suggested to play a role in the processing of emotional information. The aim of this study was to investigate whether calcineurin may be involved in the effect of antidepressants. We first found that chronic antidepressant treatment in mice leads to an increase of calcineurin levels in the hippocampus. We then studied the behavioral and molecular responses to fluoxetine of mice with a genetic overactivation of calcineurin in the hippocampus (constitutively-activated calcineurin transgenic mouse line #98, CN98 mice). We observed that CN98 mice are more sensitive to the behavioral effect of fluoxetine and desipramine tested in the tail suspension test. Moreover, the basal expression of growth factor brain-derived neurotrophic factor and subunit 1 of AMPA glutamate receptor, GluR1, both of which are modified after chronic antidepressant administration, are altered in the hippocampus of CN98 mice. These results suggest that calcineurin-dependent dephosphorylation plays an important role in the mechanisms of action of antidepressants, providing a new starting point for developing improved therapeutic treatments for depression.     

Calcium/calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain

A second isoform of Ca2+/calmodulin-dependent-kinase II inhibitor protein (CaM-KIIN) has been identified using the yeast two-hybrid screen. The 1.8kb message encodes a 78 residue CaM-KIINalpha that is 65% identical in its putative open-reading frame and 95% identical in its inhibitory domain to the previously characterized CaM-KIINbeta. CaM-KIINalpha exhibits inhibitory properties towards recombinant mouse CaM-kinase IIalpha indistinguishable from CaM-KIINbeta. The 27 amino acid inhibitory peptide (CaM-KIINtide) derived from CaM-KIIN has the ability to inhibit brain CaM-kinase II activity from multiple organisms including rat, Drosophila and goldfish. Northern analysis of various rat tissues indicates that CaM-KIINalpha is specific to brain whereas CaM-KIINbeta message is also present in testis. In situ hybridization shows a general distribution of both isoforms in rat brain with stronger localization of CaM-KIINbeta in cerebellum and hindbrain and CaM-KIINalpha in frontal cortex, hippocampus and inferior colliculus. An antibody that recognizes both isoforms shows a distribution of CaM-KIIN in rat brain that correlates with immunoreactivity of CaM-kinase II. In cultured mature hippocampal neurons, CaM-KIIN is present in cell bodies and dendrites but, unlike CaM-kinase II, does not display punctate staining at synapses. These results suggest a localized function for CaM-KIIN in inhibiting specialized pools of CaM-kinase II.