The poliovirus empty capsid specifically recognizes the poliovirus receptor and undergoes some, but not all, of the transitions associated with cell entry

Experimental results presented here demonstrate that the poliovirus empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with mature virus for binding sites on cells. Hence, induced changes in the structure and/or stability of the particle by RNA encapsidation and virus maturation are not necessary for recognition by receptor. In mature virus, heat-induced rearrangements mimic those induced by receptor at physiological temperatures in several important respects, namely, expulsion of VP4 and externalization of the VP1 N-terminal arm. It is shown here that in the empty capsid the VP1 N-terminal arm is externalized but the VP4 portion of VP0 is not. Thus, these two hallmark rearrangements associated with cell entry can be uncoupled.     

TVB-N Correlation with Odor Evaluation and Aerobic Plate Count in Mahi-Mahi (Coryphaena hippurus)

ABSTRACT: Two groups of 6 mahi-mahi fillets, one untreated and the other dipped in a suspension of Morganella morganii , were refrigerated at 7°C and sampled after 0, 2, 4, 6, 8, and 10 d of storage. Results were similar for both groups of fish. Total volatile base-nitrogen (TVB-N) reached 30 mg-N/100 g on d 3, at which time aerobic plate count (APC) reached 10⁶ colony forming units (CFU)/g and odor scores reached 6 on a scale of 1 to 10 (10 very fresh and 1 very spoiled). Good correlation existed between TVB-N and odor intensity (r = 0.84), between TVB-N and the log₁₀ APC (r = 0.71), and between odor and the log₁₀ APC (r = 0.91). These results show that TVB-N can serve as a good indicator of chilled mahi-mahi quality.     

X-ray crystal structures of the S229A mutant and wild-type MurB in the presence of the substrate enolpyruvyl-UDP-N-acetylglucosamine at 1.8-A resolution

MurB catalyzes the second committed step in the synthesis of peptidoglycan, a key component of the bacterial cell wall. The crystal structures of both a S229A mutant and wild-type MurB in the presence of the substrate enolpyruvyl-UDP-N-acetylglucosamine were solved and refined at 1.8 A resolution. The single point mutation of residue 229 from serine to alanine eliminated a hydroxyl group which has previously been proposed to play a critical role as a proton donor during the second half-reaction of MurB, namely, reoxidation of FADH2 and reduction of the enolpyruvyl substrate. The mutation also resulted in the loss of the water molecule-hydrogen bonded to the serine hydroxyl in the wild-type structure changing the hydrogen-bonding network with in the active site. Comparison of the wild-type and S229A mutant structures confirms that the dramatic kinetic defect of an approximately 10(7)-fold decrease observed for the Ser 229 Ala mutant in the second half-reaction [Benson, T.E., Walsh, C.T., & Massey, V. (1997) Biochemistry 36, 796-805] is a direct result of the loss of the serine hydroxyl moiety rather than other nonspecific active-site changes or general structural defects.     

The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: relationship to the structure of alpha-like DNA polymerases

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.     

Using Benthic Indicator Species and Community Gradients to Optimize Restoration in the Arid,Endorheic Walker River Watershed,Western USA

The challenge of restoring watersheds in arid regions often requires the development of novel scientific tools to guide management. The Walker Basin Program was created to reverse ecological decline in an arid, endorheic watershed through scientifically guided restoration. As part of this programme, 3 years of benthic macroinvertebrate samples were collected seasonally at 10 sites that represent the diversity of river environments from the high‐mountain headwaters to a desert terminal lake. Samples were analysed to quantify baseline conditions in reference and degraded reaches of river and identify opportunities and constraints for aquatic community restoration. Naturally harsh environments in the lower river characterized by high temperatures and low base flow combined with a weak understanding of reference conditions to limit the utility of commonly used indices for quantifying biotic integrity. A flexible approach was employed using a combination of indicator species analysis, cluster analysis, canonical correspondence analysis, and community tolerance indices to evaluate the variation of benthic macroinvertebrate community composition across a set of environmental gradients. Results demonstrate that benthic communities in the watershed are primarily influenced by a longitudinal gradient related to elevation. A strong secondary community gradient caused by anthropogenic nutrient loading may constrain restoration effectiveness in some parts of the watershed. Restoration activities should improve water quality conditions and initially target areas of the watershed less affected by nutrient loading. Results also demonstrate that benthic communities shift longitudinally. These shifts should be monitored to inform adaptive management of restoration actions. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.     

Free Amino Acids in Dark- and White-muscle Fish as Determined by O-phthaldialdehyde Precolumn Derivatization

ABSTRACT: Free amino acids in fish reflect microbial spoilage, and are precursors of biogenic amines, factors of health concern and indicators offish decomposition. The objective of this research was to quantify levels of free histidine, lysine, ornithine and glutamine, which may become originators of highly undesirable histamine, cadaverine and putrescine in fish tissue. Liquid chromatography using o-phthaldialdehyde (OPA) pre-column derivatization, gradient elution and a C18 column were used for separation. Histidine was higher in white tissue of mahi-mahi and tuna than in red tissue. No significant difference in lysine levels was found between the two tissues of tuna. Glutamine and ornithine were higher in red tissue of mahi-mahi and tuna. Red snapper had higher levels of lysine than histidine.     

Metallic nanohole arrays on fluoropolymer substrates as small label-free real-time bioprobes

We describe a nanoplasmonic probing platform that exploits small-dimension (相似文献