Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas

Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.     

Kinesin undergoes a 9 S to 6 S conformational transition.

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.     

福建省输入性D8基因型麻疹病毒分子流行病学特征

为分析福建省输入性D8基因型麻疹病毒分子流行病学特征,采集咽拭子标本采用实时荧光定量反转录-聚合酶链反应(Real-time RT-PCR)筛查麻疹病毒核酸.用反转录-聚合酶链反应(RT-PCR)扩增麻疹病毒核酸筛查阳性咽拭子及Vero/Slam细胞培养阳性产物,对麻疹病毒核蛋白(Nucleoprotein,N)羧基(COOH)端634个核苷酸(Nucleotides,nt)片段进行测序分析,构建系统进化树.最终分离获得1株麻疹病毒株,26条麻疹病毒N蛋白羧基末端450个nt序列.亲缘性分析发现,所有福建麻疹毒株与WHOD8基因型参考株(MVi/Manchester.GBR/30.94)在亲缘关系树上同属一个大分支,两者核苷酸序列和氨基酸(Amino acid,aa)序列同源性分别为96.4％～99.1％和96.7％～98.0％.其中2014年的福建毒株 MVs/Fujian.CHN/28.14和 MVs/Fujian.CHN/30.14与越南胡志明市2014年分离株MVs/HoChiMinh.VNM/11.14及美国纽约2013年分离株MVs/New.York.USA/19.13的nt同源性为 100％;2019年的毒株MVs/Fujian.CHN/25.19与泰国龙仔厝府2018年分离株MVs/Samut.Sakhon.THA/8.18的nt同源性为100％;而剩余的23个监测毒株则与日本神户2019年分离株MVs/KobeC.JPN/28.19的核苷酸同源性和氨基酸同源性最高,分别为99.6％～100.0％和99.3％～100.0％.在病毒N蛋白羧基端150个氨基酸位点上,福建株与WHO D8参考株存在3～5个氨基酸位点差异.而与现用的疫苗株(Shanghai-191)相比,存在17～19个氨基酸变异位点,其中有14个氨基酸位点为所有福建株共有的变异位点,这些位点的变异总体上未对编码蛋白的氨基酸造成明显改变.结论是福建省成功分离获得1株D8基因型麻疹毒株.D8基因型为福建省发现的输入性麻疹基因型,病毒N蛋白羧基端氨基酸位点上与疫苗株相比均出现了差异位点.     

Effect of pH on infectivity and morphology of ecotropic moloney murine leukemia virus

A number of factors affect the infectivity of retroviruses. The effect of pH on infectivity and morphology of ecotropic moloney murine leukemia virus (MoMuLV) was determined in this work. The ecotropic MoMuLVs were found to remain infectious at a narrow pH range from 5.5 to 8.0. Our experiments indicated that the viruses were inactivated swiftly at lower or higher pH. Within 5 min of exposure to pH 4 about 95% of the viruses lost infectiousness. The viruses were completely inactivated after exposure to pH < 3 or pH >11 for 5 min. The inactivation of MoMuLV was irreversible. Electron microscopy revealed that ecotropic MoMuLV remained round-shaped at pH between 7.0 and 5. They became irregular with a convex head at pH < 4. At pH 2, virtually all virion particles were penetrated by stains, causing the accumulation of heavy metals inside the particles. The penetration of heavy metal inside the particles indicated the disassociation of the lipid bilayer of the viruses at low pH. A FACS-based screening strategy for selecting high-titer retrovirus producing cell lines is also presented in this report.     

Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells

The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogen-activated protein kinase kinase 1/2, prevented the VPA-induced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvation-induced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.     

New Insight into Ni‐Rich Layered Structure for Next‐Generation Li Rechargeable Batteries

An increase in the amount of nickel in LiMO₂ (M = Ni, Co, Mn) layered system is actively pursued in lithium‐ion batteries to achieve higher capacity. Nevertheless, fundamental effects of Ni element in the three‐component layered system are not systematically studied. Therefore, to unravel the role of Ni as a major contributor to the structural and electrochemical properties of Ni‐rich materials, Co‐fixed LiNi_0.5+xCo_0.2Mn_0.3–xO₂ (x = 0, 0.1, and 0.2) layered materials are investigated. The results, on the basis of synchrotron‐based characterization techniques, present a decreasing trend of Ni²⁺ content in Li layer with increasing total Ni contents. Moreover, it is discovered that the c_hex.‐lattice parameter of layered system is not in close connection with the interslab thickness related to actual Li ion pathway. The interslab thickness increases with increasing Ni concentration even though the c_hex.‐lattice parameter decreases. Furthermore, the lithium ion pathway is preserved in spite of the fact that the c‐axis is collapsed at highly deintercalated states. Also, a higher Ni content material shows better structural properties such as larger interslab thickness, lower cation disorder, and smoother phase transition, resulting in better electrochemical properties including higher Li diffusivity and lower overpotential when comparing materials with lower Ni content.     

Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment

Using both electron microscopy and immunological methods, we have characterized a number of changes occurring in rat fibroblasts after heat-shock treatment. Incubation of the cells for 3 h at 42 degrees-43 degrees C resulted in a number of changes within the cytoplasm including: a disruption and fragmentation of the Golgi complex; a modest swelling of the mitochondria and subtle alterations in the packing of the cristae; and alterations in cytoskeletal elements, specifically a collapse and aggregation of the vimentin-containing intermediate filaments around the nucleus. A number of striking changes were also found within the nuclei of the heat-treated cells: (a) We observed the appearance of rod-shaped bodies consisting of densely packed filaments. Using biochemical and immunological methods, these nuclear inclusion bodies were shown to be comprised of actin filaments. (b) Considerable alterations in the integrity of the nucleoli were observed after the heat-shock treatment. Specifically, there appeared to be a general relaxation in the condensation state of the nucleoli, changes in both the number and size of the granular ribonucleoprotein components, and finally a reorganization of the nucleolar fibrillar reticulum. These morphological changes in the integrity of the nucleoli are of significant interest since previous work as well as studies presented here show that two of the mammalian stress proteins, the major stress-induced 72-kD protein and the 110-kD protein, localize within the nucleoli of the cells after heat-shock treatment. We discuss these morphological changes with regards to the known biological and biochemical events that occur in cells after induction of the stress response.