Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins.

This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE. Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames. We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors. Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions. Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells. An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complex formation is necessary for efficient processing of the gE precursor to its mature form. One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse. Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.     

Megafauna extinction,tree species range reduction,and carbon storage in Amazonian forests

During the Late Pleistocene and early Holocene 59 species of South American megafauna went extinct. Their extinction potentially triggered population declines of large‐seeded tree species dispersed by the large‐bodied frugivores with which they co‐evolved, a theory first proposed by Janzen and Martin (1982). We tested this hypothesis using species range maps for 257 South American tree species, comparing 63 species thought to be primarily distributed by megafauna with 194 distributed by other animals. We found a highly significant (p < 0.001) decreased mean range size of 26% for the megafauna dispersed fruit (n = 63 species) versus fruit dispersed by other animals (n = 194), results which support the hypothesis. We then developed a mathematical model of seed dispersal to estimate the theoretical impact of megafauna extinction on tree species range and found the estimated dispersal capacity (Φ_seed) of a 2 g seed decreases by > 95% following disperser extinction. A numerical gap dynamic simulations suggests that over a 10 000 yr period following the disperser extinctions, the average convex hull range size of large‐seeded tree species decreased by ～ 31%, while the estimated decrease in population size was ～ 54%, indicating a likely greater decrease in species population size than indicated by the empirical range patterns. Finally, we found a positive correlation between seed size and wood density of animal‐dispersed tree species implying that the Late Pleistocene and early Holocene megafaunal extinctions reduced carbon content in the Amazon by ～ 1.5 ± 0.7%. In conclusion, we 1) provide some empirical evidence that megafauna distributed fruit species have a smaller mean range size than wind, water or other animal‐dispersed species, 2) demonstrate mathematically that such range reductions are expected from megafauna extinctions ca 12 000 yr ago, and 3) illustrate that these extinctions may have reduced the Amazon's carbon storage capacity.     

A network approach for inferring species associations from co‐occurrence data

Positive and negative associations between species are a key outcome of community assembly from regional species pools. These associations are difficult to detect and can be caused by a range of processes such as species interactions, local environmental constraints and dispersal. We integrate new ideas around species distribution modeling, covariance matrix estimation, and network analysis to provide an approach to inferring non‐random species associations from local‐ and regional‐scale occurrence data. Specifically, we provide a novel framework for identifying species associations that overcomes three challenges: 1) correcting for indirect effects from other species, 2) avoiding spurious associations driven by regional‐scale distributions, and 3) describing these associations in a multi‐species context. We highlight a range of research questions and analyses that this framework is able to address. We show that the approach is statistically robust using simulated data. In addition, we present an empirical analysis of > 1000 North American tree communities that gives evidence for weak positive associations among small groups of species. Finally, we discuss several possible extensions for identifying drivers of associations, predicting community assembly, and better linking biogeography and community ecology.     

Separation and quantitation of (R)- and (S)-atenolol in human plasma and urine using an alpha 1-AGP column

A method for the determination of (R)- and (S)-atenolol in human plasma and urine is described. The enantiomers of atenolol are extracted into dichloromethane containing 3% heptafluorobutanol followed by acetylation with acetic anhydride at 60 degrees C for 2 h. The acetylated enantiomers were separated on a chiral alpha 1-AGP column. Quantitation was performed using fluorescence detection. A phosphate buffer pH 7.1 (0.01 M phosphate) containing 0.25% (v/v) acetonitrile was used as mobile phase. The described procedure allows the detection of less than 6 ng of each enantiomer in 1 ml plasma. The relative standard deviation is 4.4% at 30 ng/ml of each enantiomer in plasma. The plasma concentration of (R)- and (S)-atenolol did not differ significantly in two subjects who received a single tablet of racemic atenolol. The R/S ratio of atenolol in urine was approximately 1.     

Replacement of the pseudorabies virus glycoprotein gIII gene with its postulated homolog, the glycoprotein gC gene of herpes simplex virus type 1.

gIII, the major envelope glycoprotein of pseudorabies virus (PRV), shares approximately 20% amino acid similarity with glycoprotein gC of herpes simplex virus type 1 (HSV-1) and HSV-2. We describe here our first experiments on the potential conservation of function between these two genes and gene products. We constructed PRV recombinants in which the gIII gene and regulatory sequences have been replaced with the entire HSV-1 gC gene and its regulatory sequences. The gC promoter functions in the PRV genome, and authentic HSV-1 gC protein is produced, albeit at a low level, in infected cells. The gC protein is present at the cell surface but cannot be detected in the PRV envelope.     

The gIII glycoprotein of pseudorabies virus is involved in two distinct steps of virus attachment.

The entry of herpesviruses into cells involves two distinct stages: attachment or adsorption to the cell surface followed by internalization. The virus envelope glycoproteins have been implicated in both stages. Pseudorabies virus attaches to cells by an early interaction that involves the viral glycoprotein gIII and a cellular heparinlike substance. We examined the role of gIII in the attachment process by analysis of a set of viruses carrying defined gIII mutations. The initial attachment of gIII mutants with an internal deletion of 134 amino acids (PrV2) to MDBK cells was indistinguishable from that of wild-type virus. The adsorption of these mutants was, however, much more sensitive than that of wild-type virus to competing heparin. Furthermore, while attachment of wild-type virus to MDBK cells led to a rapid loss of sensitivity to heparin, this was not the case with PrV2, which could be displaced from the cell surface by heparin after it had attached to the cells. We conclude that glycoprotein gIII is involved in two distinct steps of virus attachment and that the second of these steps but not the first is defective in PrV2.