Inactivation of murine leukemia virus by compounds that react with the zinc finger in the viral nucleocapsid protein.

All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two copies of the conserved sequence motif C-X2-C-X4-H-X4-C. The conserved cysteine and histidine residues coordinate a zinc ion in each such motif. Rice et al. (W. G. Rice, J. G. Supko, L. Malspeis, R. W. Buckheit, Jr., D. Clanton, M. Bu, L. Graham, C. A. Schaeffer, J. A. Turpin, J. Domagala, R. Gogliotti, J. P. Bader, S. M. Halliday, L. Coren, R. C. Sowder II, L. 0. Arthur, and L. E. Henderson, Science 270:1194-1197, 1995) have described a series of compounds which inactivate human immunodeficiency virus type 1 (HIV-1) particles and oxidize the cysteine thiolates in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. The killed MuLV particles were found to be incapable of synthesizing full-length viral DNA upon infection of a new host cell. When MuLV particles are synthesized in the presence of one of these compounds, the normal maturational cleavage of the Gag polyprotein does not occur. The compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV- I by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses and the lethality of all known mutations altering the zinc-binding residues suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.     

Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus gluatamate dehydrogenase

Abstract: The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus has been completed at 2.2 Å resolution. The structure has been compared with the glutamate dehydrogenases from the mesophiles Clostridium symbiosum, Escherichia coli and Neurospora crassa . This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues. Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited. The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures.     

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.     

Biofilm ecology: On-line methods bring new insights into mic and microbial biofouling

Microbial biofilms were formed on coupons with defined coatings in once-through laminar flow fields of controlled bulk-phase composition and shear. Dilute media were utilized to select for biofilm growth. The formation, succession, and stability of the biofilms were monitored with non-destructive on-line methods (fluorescence, bioluminescence, attenuated total reflectance Fourier transform infrared spectrometry [ATR-FTIR] and electrochemical impedance spectroscopy) and by high resolution destructive analysts (viable and direct counts and phospholipid fatty acid signature methods) at the termination of the experiments. Biofilms of reproducible composition can be formed and the order of inoculation of multi-component biofilms affects their composition at harvest. The corrosion rates of mild steel depended on the biofilm composition but not the attached biomass. Examination of biofilms with the scanning vibrating electrode in a microscope field showed effects of heterogeneity in biofilm structure which promoted localized anodic activity. Pseudomonas stains were engineered to contain the lux gene cassette as a "reporter"; and the formation of the exopolymer alginate was shown not to promote attachment of the strain or secondary colonization by Vibrio. Examination of mutants forming different alginate structures showed differential attachment and biofilm structure. Studies of mutants of lipopolysaccharide structure showed differential attachment to substrata. Specific antifouling and fouling-release coatings showed a wide range of attachment and release properties as well as sublethal toxicity.     

Effects of external stimuli on environmental bacterial strains harboring analgD-lux bioluminescent reporter plasmid for the study of corrosive biofilms

An alginic acid biosynthesis bioluminescent reporter plasmid, pUTK50, was transconjugated into environmental strains ofPseudomonas putida, Pseudomonas fluorescens, andStenotrophomonas maltophilia. Bioluminescent transconjugates were selected from each strain for investigation of environmental stress factors that promote alginic acid exopolymer biosynthesis in developing biofilms. Environmental stimuli associated with increased levels of alginate synthesis, in a previously developed organism,P. aeruginosa FRD1, were applied to the environmental strains. Increased salt concentrations and higher ratios of nitrate vs ammonium ions as the limiting nitrogen source induced bioluminescence in FRD1 and the environmental strains. However, for environmental strains ofP. putida, P. fluorescens andS. maltophilia, polysaccharides were detected with low uronic acids content and different structural components. When tested within a biofilm,S. maltophilia O46 demonstrated exceptional adhesive and corrosive properties while alginic acid synthesis was not high. In most of the environmental strains, periods of increased bioluminescence were induced by external stimuli, but exopolysaccharides other than alginic acid were expressed. It is hypothesized that the environmental strains have homologous but nonidentical promoter sequences which are responsive to certain environmental stimuli and may control genes necessary for the production of alternative exopolysaccharides.     

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

豚鼠主动脉前庭组织的自律性研究和形态学观察

用细胞内微电极技术,对８２只豚鼠主动脉自发电活动进行观察和分析,表明,１６例（１９．５％）出现自律性电活动;６１例（７４．４％）在电刺激诱导下也可出现自律性电活动。进一步观察发现,在主动脉瓣右瓣与后瓣交界以下的局部区域,可记录到有明显４相自动增极化的慢反应自律细胞样的动作电位,并可被钙通道阻滞剂Ｍｎ＾２＋,硝苯吡啶阻断。这一部位之外的其它大部分区域均表现为快反应动作电位,经光镜和电镜观察（ｎ＝１３     

Sindbis virus attachment: isolation and characterization of mutants with impaired binding to vertebrate cells.

Sindbis virus can infect a broad range of insect and vertebrate cell types. The ability to restrict tissue tropism and target virus infection to specific cell types would expand the usefulness of engineered alphaviruses as gene expression vectors. In this study, virus pools derived from libraries of full-length Sindbis virus cDNA clones containing random insertion mutations in the PE2 or E1 virion glycoprotein gene were screened for mutants defective for binding to vertebrate cells. Binding-competent mutants were depleted by serial adsorption to chicken embryo fibroblast (CEF) monolayers at 4 degrees C, and the remaining population was amplified by immune-enhanced infection of P388D1 cells. From the PE2 libraries, 12 candidate mutants showing reduced cytopathic effects on CEF monolayers were isolated and three representative mutants, NB1, NB2, and NB12, were characterized in detail. Insertion mutations for NB1 and NB12 were found near the PE2 cleavage site, whereas the insertion in NB2 occurred between residues 69 and 74 of E2. Although virion assembly and release occurred normally for all three mutants, PE2 cleavage was completely (NB1) or partially (NB12) blocked for the mutants with insertions near the PE2 cleavage site. Both NB1 and NB2 were defective for binding to CEF and BHK-21 cells. Mild trypsin digestion of isolated NB1 virions resulted in PE2 cleavage and partially restored binding to CEF. Besides defective binding, NB1 also exhibited slower CEF penetration kinetics. Consistent with previous work, these results implicate PE2 cleavage and domains in the N-terminal portion of E2 as important determinants of alphavirus binding and penetration. Binding-defective mutants such as NB2, which exhibit normal particle assembly, release, and penetration, may be useful for future efforts to target Sindbis virus infection.