AcMNPV核衣壳的形态发生

报道了缺失多角体蛋白基因的AcMNPV在sf9细胞内核衣壳的形态发生过程。病毒衣壳蛋白首先装配成许多呈束状排列的直径为34nm中空长管状结构,然后是病毒DNA进入管内,装有DNA的长管按一定的长度间隔断开,形成成束的核衣壳,每个核衣壳的大小约34×260nm,最后成束的核衣壳被囊膜包被形成完整的多粒包埋型病毒粒子。     

Purification and characterization of a Mn2+/phospholipid-dependent protein phosphatase from pig brain membranes

A Mn²⁺/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase theV _max of the phosphatase but had no effect on theK _m value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn²⁺. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn²⁺ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn²⁺/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn²⁺ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn²⁺ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase.     

A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function

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Long-term daily access to alcohol alters dopamine-related synthesis and signaling proteins in the rat striatum

Chronic alcohol exposure can adversely affect neuronal morphology, synaptic architecture and associated neuroplasticity. However, the effects of moderate levels of long-term alcohol intake on the brain are a matter of debate. The current study used 2-DE (two-dimensional gel electrophoresis) proteomics to examine proteomic changes in the striatum of male Wistar rats after 8 months of continuous access to a standard off-the-shelf beer in their home cages. Alcohol intake under group-housed conditions during this time was around 3–4 g/kg/day, a level below that known to induce physical dependence in rats. After 8 months of access rats were euthanased and 2-DE proteomic analysis of the striatum was conducted. A total of 28 striatal proteins were significantly altered in the beer drinking rats relative to controls. Strikingly, many of these were dopamine (DA)-related proteins, including tyrosine hydroxylase (an enzyme of DA biosynthesis), pyridoxal phosphate phosphatase (a co-enzyme in DA biosynthesis), DA and cAMP regulating phosphoprotein (a regulator of DA receptors and transporters), protein phosphatase 1 (a signaling protein) and nitric oxide synthase (which modulates DA uptake). Selected protein expression changes were verified using Western blotting. We conclude that long-term moderate alcohol consumption is associated with substantial alterations in the rat striatal proteome, particularly with regard to dopaminergic signaling pathways. This provides potentially important evidence of major neuroadaptations in dopamine systems with daily alcohol consumption at relatively modest levels.     

Interaction between the C‐terminal domains of measles virus nucleoprotein and phosphoprotein: A tight complex implying one binding site

The intrinsically disordered C‐terminal domain (N_TAIL) of the measles virus (MeV) nucleoprotein undergoes α‐helical folding upon binding to the C‐terminal X domain (XD) of the phosphoprotein. The N_TAIL region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489–506. In the previous studies published in this journal, we obtained experimental evidence supporting a K_D for the N_TAIL–XD binding reaction in the nM range and also showed that an additional N_TAIL region (Box3, aa 517–525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (K_D in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re‐evaluate the role of Box3 in N_TAIL–XD binding. Since our previous studies relied on N_TAIL‐truncated forms possessing an irrelevant Flag sequence appended at their C‐terminus, we, herein, generated an N_TAIL devoid of Box3 and any additional C‐terminal residues, as well as a form encompassing only residues 482–525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N_TAIL forms. Results effectively argue for the presence of a single XD‐binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high‐affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point.     

肾移植术后巨细胞病毒pp65检测的临床意义

目的探讨肾移植受者术后,巨细胞病毒被膜磷蛋白pp65的检测在活动性巨细胞病毒感染中的意义。方法用间接免疫荧光法检测肾移植受者术后HCMV-pp65,同时用酶联免疫捕获法检测HCMV-IgM抗体,共采集91份血标本。结果 91份血标本中,HCMV-pp65阳性24份（26.4%）,HCMV-IgM抗体阳性4份（4.4%）,在24例HC-MV-pp65抗原血症阳性的患者中,有20例出现HCMV感染症状及HCMV病。结论 HCMV-pp65在活动性巨细胞病毒感染的检测中具有早期、准确的优点,可辅助临床对HCMV感染进行早期诊断与治疗。     

Spectrin interacts with EVL (Enabled/vasodilator-stimulated phosphoprotein-like protein), a protein involved in actin polymerization

BACKGROUND INFORMATION: The alpha- and beta-spectrin chains constitute the filaments of the spectrin-based skeleton, which was first identified in erythrocytes. The discovery of analogous structures at plasma membranes of eukaryotic cells has led to investigations of the role of this spectrin skeleton in many cellular processes. The alphaII-spectrin chain expressed in nucleated cells harbours in its central region several functional motifs, including an SH3 (Src homology 3) domain. RESULTS: Using yeast two-hybrid screening, we have identified EVL [Enabled/VASP (vasodilator-stimulated phosphoprotein)-like protein] as a new potential partner of the alphaII-spectrin SH3 domain. In the present study, we investigated the interaction of the alphaII-spectrin SH3 domain with EVL and compared this with other proteins related to EVL [Mena (mammalian Enabled) and VASP]. We confirmed the in vitro interaction between EVL and the alphaII-spectrin SH3 domain by GST (glutathione S-transferase) pull-down assays, and showed that the co-expression of EVL with the alphaII-spectrin SH3 domain in COS-7 cells resulted in the partial delocalization of the SH3 domain from cytoplasm to filopodia and lamellipodia, where it was co-localized with EVL. In kidney epithelial and COS-7 cells, we demonstrated the co-immunoprecipitation of the alphaII-spectrin chain with over-expressed EVL. Immunofluorescence studies showed that the over-expression of EVL in COS-7 cells promoted the formation of filopodia and lamellipodia, and the expressed EVL was detected in filopodial tips and the leading edge of lamellipodia. In these cells over-expressing EVL, the alphaII-spectrin membrane labelling lagged behind EVL staining in lamellipodia and filopodia, with co-localization of these two stains in the contact area. In kidney epithelial cell lines, focused co-localization of spectrin with expressed EVL was observed in the membrane of the lateral domain, where the cell-cell contacts are reinforced. CONCLUSIONS: The possible link between the spectrin-based skeleton and actin via the EVL protein suggests a new way of integrating the spectrin-based skeleton in areas of dynamic actin reorganization.     

HLA-A*0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins

The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla_bind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be the first identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-gamma stimulation of blood CD8+ T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.     

血管扩张刺激磷蛋白在细胞骨架调节中的作用

细胞骨架动力学的调节在细胞粘附、细胞变形、细胞移动等生理过程中是必需的。血管扩张刺激磷蛋白（vasodilator-stimulated phosphoprotein,VASP）是一种肌动蛋白结合蛋白。该蛋白包含以下结构域：EVH1（Ena／VASP homolog1）区、EVH2（Ena／VASP homolog2）区及PRR（proline—rich regions）区。近年来,研究发现VASP在与细胞骨架调节有关的各种细胞行为中起着重要作用,如神经细胞轴索的延伸、T细胞的移动、成纤维细胞的迁移等。VASP的磷酸化受PKG（cGMP-dependent protein kinase）和PKA（cAMP—dependent protein kinase）的调控。在粘附斑的形成与脱落过程中,该磷酸化起着一个“开关”的作用。本文将就近20年来VASP的研究成果,特别是近年来的进展情况做一综述。