Non-random Escape Pathways from a Broadly Neutralizing Human Monoclonal Antibody Map to a Highly Conserved Region on the Hepatitis C Virus E2 Glycoprotein Encompassing Amino Acids 412–423

A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412–423 is conserved and antibodies to 412–423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412–423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412–423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which can co-exist with lower potency or levels of neutralizing activities.     

Preliminary Study on Development,Germination and Desiccation Tolerance of Jatropha curcas (Euphorbiaceae)

The developmental changes in morphology and germinability of Jatropha curcas seeds, effects of temperature and light on seed germination , and changes in desiccation tolerance of mature seeds were studied in this paper. The results indicated that J. curcas seeds reached a physiological maturation at 58 days after anthesis, and that seed germination percentage reached a peak at physiological maturation, and then decreased. The optimal germination temperature was 25 to 30℃. J. curcas seeds were insensitive to dehydration at physiological maturation . There was not a notable effect of light on seed germination. Therefore, J. curcas seed was a non-photoblastic and orthodox seeds .     

Vertical Migration and Mortality of Marine Benthos in Dredged Material: A Synthesis

This account describes the comparative response of four species of benthic invertebrates to burial in terms of vertical migration and mortality, and provides a synthesis of studies and recommendations upon which to assess future impacts. The species featured were the bivalve Mercenaries mercenaria, the amphipod crustacean Parahaustorius longimerus, and the polychaetes Scoloplos fragilis and Nereis succinea. There was evidence of synergistic effects on burrowing activity and mortality with changes in time of burial, sediment depth, sediment type and temperature. In sediment with silt-clay, N. succinea was the most resistant species followed by M. mercenaria, S. fragilis and P. longimerus. In sediment without silt-clay the order of percent mortality was reversed. Studies of surface water chemistry and sediment geochemistry showed that dissolved oxygen decreased significantly and ammonia and sulfide increased significantly between the surface and below 2.0 cm within a 15-day period. Based on these results and physiological tolerances from the literature it was concluded that M. mercenaria and N. succinea would be relatively resistant to chemical effects of spoil disposal, whereas S. fragilis and P. longimerus would be less resistant to such effects. Vertical migration of benthic invertebrates through dredge disposal can be a viable mechanism of recolonization under certain conditions. Some effects of burial of benthos can be predicted based on morphology, behavior and physiology. These biological features were discussed with examples dealing with molluscs, crustaceans, and polychaetes. Finally, recommendations were made concerning the type of studies to provide additional data to aid management agencies in decision making about future dredging and disposal practices.     

Selective effects of gold (III) on parathyroid hormone-, prostaglandin E2- and guanine nucleotide-sensitive adenylate cyclase

Gold(III) (Au(III)) up to 0.25 microM increased parathyroid hormone- and prostaglandin E2-sensitive chick osteoblast adenylate cyclase activity without affecting 5'-guanylylimidodiphosphate-stimulated enzyme activity. Au(III) at 5-50 microM inhibited hormone- and nucleotide-mediated activation of adenylate cyclase. Basal adenylate cyclase activity was not influenced by Au(III) in the given concentrations. Treatment of membranes with 5'-guanylylimidodiphosphate prior to incubation with Au(III) prevented the inhibitory effect of Au(III) on adenylate cyclase. Our data suggest that Au(III) alters the response of adenylate cyclase to agonists most likely through interaction with specific sulfhydryl groups associated with the enzyme system.     

Comparative effects of dopamine and dobutamine on glucoregulation in a rat model.

The influence of dopamine as compared with dobutamine on glucose homeostasis has been assessed in thyroidectomized euthyroid rats. Both sympathomimetic agents were given intravenously over 6 h at four dosages, varying from 2 to 30 micrograms.kg-1.min-1. Immediately before the end of the infusion period, serum concentrations of glucose and insulin as well as plasma glucagon concentrations were measured. Dobutamine infusions did not exert any influence on these parameters. At a dose of 7.5 micrograms.kg-1.min-1, dopamine infusion caused a decrease in glucose concentrations, accompanied by a rise of glucagon and insulin levels. Glucose levels were significantly increased in the presence of unaltered insulin and decreasing glucagon levels at higher dopamine doses. The rise in glucose levels was reversed by 8 micrograms.kg-1.min-1 and inverted to a decrease by 12 micrograms.kg-1.min-1 of the alpha-adrenergic blocking agent phentolamine, simultaneously infused with 15 micrograms.kg-1.min-1 dopamine, while the insulin levels were increased and glucagon levels remained elevated. These findings demonstrate that dopamine acts on glucoregulation divergently, according to the dosage applied. The data suggest that dopamine rather than dobutamine treatment may disturb glucose homeostasis.     

Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins.     

Photochemical characteristics in a soybean mutant

Chloroplasts were isolated from wild type (DG) and heterozygous mutant (LG) soybean (Glycine max) leaves, and various biochemical functions were compared. Noncyclic electron transport, and its coupled phosphorylation, cyclic phosphorylation and H⁺ ion transport in both systems, were 3 to 5 times faster in rate (on a chlorophyll basis) in the mutant plastids. On a chloroplast lamellar protein basis, the mutant plastid rates were 1.5 to 2.5 times the wild type rates.     

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.