Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350

The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK.     

Foreleg Autotomy Reduces Mating Success of Male <Emphasis Type="Italic">Schizocosa ocreata</Emphasis> Wolf Spiders

Autotomy is a taxonomically widespread antipredator tactic that allows animals to escape life-threatening situations. Opposing the benefits of survival, animals that have autotomized appendages may later suffer reduced ability in important determinants of fitness. Male Schizocosa ocreata wolf spiders use their forelegs during courtship for visual displays, for tactile courtship, and to defend against attacks by females. In nature they are often found missing one, and sometimes both, forelegs. We found that autotomy of one foreleg has little effect on male ability to mate with virgin females, but that autotomy of both forelegs causes a significant reduction in mating success. Among males that mated, autotomy of one or both forelegs did not influence latency until mating, period spent mounted, probability that his mate would accept a subsequent suitor, or probability that his mate would kill a subsequent suitor.     

Genetic variants in the 8q24 locus and risk of testicular germ cell tumors

Much evidence supports the premise that population genetic variation contributes significantly to the risk of testicular germ-cell tumor (TGCT). However, investigations of the association between genomic markers and TGCT susceptibility are scarce. Single nucleotide polymorphisms (SNPs) at the locus 8q24 have recently been found to be associated with prostate, colorectal and breast cancer. The US Servicemen’s testicular tumor environmental and endocrine determinants (STEED) study was used to investigate the association of 15 specific 8q24 SNPs with TGCT and its two main histologic groups of seminoma and nonseminoma. Conditional and unconditional logistic regression models, adjusted for the matching variables of year of birth, race/ethnicity and serum date, were utilized to produce odds ratios (OR) and 95% confidence intervals (95%CI). The analysis included 680 controls and 568 TGCT cases. In the TGCT analysis, no SNP was associated with risk in both heterozygotes and variant homozygotes. When stratified by histology the seminoma analysis also showed no association with the 8q24 SNPs. Conversely, the analysis of nonseminomas had three tentative associations (rs6470494, OR_{genotype AG} = 1.15, 95%CI: 0.86–1.54; OR_{genotype GG} = 1.68, 95%CI: 1.04–2.73; p for trend = 0.04) (rs13254738, OR_{genotype GT} = 1.04, 95%CI: 0.77–1.40; OR_{genotype TT} = 1.62, 95%CI: 1.06–2.49; p for trend = 0.07) (rs10505476, OR_{genotype CT} = 0.67, 95%CI: 0.50, 0.91; OR_{genotype TT} = 0.81, 95%CI: 0.47–1.39; p for trend = 0.04). There was no linkage disequilibrium between any of the 8q24 SNPs analyzed in this population. In conclusion, this study has found little evidence for an association between the reported 8q24 SNPs and TGCTs, although the findings for nonseminoma may be worth further investigation.     

Development of methods for the genetic manipulation of <Emphasis Type="Italic">Flavobacterium columnare</Emphasis>

Background  

Flavobacterium columnare is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the Bacteroidetes, including members of the genus Flavobacterium, have been described, but these methods were not adapted to work with F. columnare.     

Limits to genetic bottlenecks and founder events imposed by the Allee effect

The Allee effect can result in a negative population growth rate at low population density. Consequently, populations below a minimum (critical) density are unlikely to persist. A lower limit on population size should constrain the loss of genetic variability due to genetic drift during population bottlenecks or founder events. We explored this phenomenon by modeling changes in genetic variability and differentiation during simulated bottlenecks of the alpine copepod, Hesperodiaptomus shoshone. Lake surveys, whole-lake re-introduction experiments and model calculations all indicate that H. shoshone should be unlikely to establish or persist at densities less than 0.5–5 individuals m⁻³. We estimated the corresponding range in minimum effective population size using the distribution of habitat (lake) sizes in nature and used these values to model the expected heterozygosity, allelic richness and genetic differentiation resulting from population bottlenecks. We found that during realistic bottlenecks or founder events, >90% of H. shoshone populations in the Sierra Nevada may be resistant to significant changes in heterozygosity or genetic distance, and 70–75% of populations may lose <10% of allelic richness. We suggest that ecological constraints on minimum population size be considered when using genetic markers to estimate historical population dynamics.     

Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics

Background: The evolution and biology of rDNA have interested biologistsfor many years, in part, because of two intriguing processes:(1) nucleolar dominance and (2) sequence homogenization. Wereview patterns of evolution in rDNA in the angiosperm genusNicotiana to determine consequences of allopolyploidy on theseprocesses. Scope: Allopolyploid species of Nicotiana are ideal for studying rDNAevolution because phylogenetic reconstruction of DNA sequenceshas revealed patterns of species divergence and their parents.From these studies we also know that polyploids formed overwidely different timeframes (thousands to millions of years),enabling comparative and temporal studies of rDNA structure,activity and chromosomal distribution. In addition studies onsynthetic polyploids enable the consequences of de novo polyploidyon rDNA activity to be determined. Conclusions: We propose that rDNA epigenetic expression patterns establishedeven in F₁ hybrids have a material influence on the likely patternsof divergence of rDNA. It is the active rDNA units that arevulnerable to homogenization, which probably acts to reducemutational load across the active array. Those rDNA units thatare epigenetically silenced may be less vulnerable to sequencehomogenization. Selection cannot act on these silenced genes,and they are likely to accumulate mutations and eventually beeliminated from the genome. It is likely that whole silencedarrays will be deleted in polyploids of 1 million years of ageand older.     

Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain

Polycomb group (PcG) proteins are required for maintaining cell identity and stem cell self-renewal. RING1B and Polycomb (Pc) are two components of a multiprotein complex called polycomb repression complex 1 (PRC1) that is essential for establishing and maintaining long-term repressed gene states. Here we characterize the interaction between the C-terminal region of RING1B (C-RING1B) and the Pc cbox domain. The C-RING1B-cbox interaction displays a 1:1 stoichiometry with dissociation constants ranging from 9.2 to 180 nM for the different Pc orthologues. NMR analysis of C-RING1B alone reveals line broadening. However, when it is in complex with the cbox domain, there is a striking change to the NMR spectrum indicative of conformational tightening. This conformational change may arise from the organization of the C-RING1B subdomains. The C-terminal regions of all PcG RING1 proteins are composed of two stretches of conserved sequences separated by a variable linker sequence. While the entire C-RING1B region is required for cbox binding, the N- and C-terminal halves of C-RING1B can be separated and are able to interact, suggesting the presence of an intramolecular interaction within C-RING1B. The flexibility within the C-RING1B structure allowing transitions between the intramolecular bound and unbound states may cause the broadened peaks of the C-RING1B NMR spectrum. Binding the cbox domain stabilizes C-RING1B, whereby broadening is eliminated. The presence of flexible regions could allow C-RING1B to bind a variety of different factors, ultimately recruiting RING1B and its associated PcG proteins to different genomic loci.     

Artesunate: developmental toxicity and toxicokinetics in monkeys

BACKGROUND: The developmental toxicity, toxicokinetics, and hematological effects of the antimalarial drug, artesunate, were previously studied in rats and rabbits and have now been studied in cynomolgus monkeys. METHODS: Groups of up to 15 pregnant females were dosed on Gestation Days (GD) 20–50 or for 3–7‐day intervals. RESULTS: At 30 mg/kg/day, 6 embryos died between GD30 and GD40. Histologic examination of 3 live embryos (GD26–GD36) revealed a marked reduction in embryonic erythroblasts and cardiomyopathy. At 12 mg/kg/day, 6 embryos died between GD30 and GD45. Four surviving fetuses examined on GD100 had no malformations, but long bone lengths were slightly decreased. At the developmental no‐adverse‐effect‐level (4 mg/kg/day), maternal plasma AUC was 3.68 ng.h/mL for artesunate and 6.93 ng.h/ml for its active metabolite, dihydroartemisinin (DHA). No developmental toxicity occurred with administration of 12 mg/kg/day for 3 or 7 days, GD29–31 or GD27–33 (maternal plasma AUC of 9.84 ng.h/mL artesunate and 16.4 ng.h/mL DHA). Exposures at embryotoxic doses were substantially lower than human therapeutic exposures. However, differences in monkey and human Vss for artesunate (0.5 L/kg vs. 0.18 L/kg) confound relying solely on AUC for assessing human risk. Decreases in reticulocyte count occur at therapeutic doses in humans. Changes to reticulocyte counts at embryotoxic doses in monkeys (≥12 mg/kg/day) were variable and generally minor. CONCLUSIONS: Artesunate was embryolethal at ≥12 mg/kg/day when dosed for at least 12 days at the beginning of organogenesis, but not when dosed for 3 or 7 days, indicating that developmental toxicity of artesunate is dependent upon duration of dosing in cynomologus monkeys. Birth Defects Res (Part B) 83:418–434, 2008. © 2008 Wiley‐Liss, Inc.     

Inter-flavin electron transfer in cytochrome P450 reductase - effects of solvent and pH identify hidden complexity in mechanism

This study on human cytochrome P450 reductase (CPR) presents a comprehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. pH-dependent redox potentiometry revealed that the thermodynamic equilibrium between various two-electron reduced enzyme species (FMNH*,FADH*; FMN,FADH2; FMNH2,FAD) is independent of pH. No shift from the blue, neutral di-semiquinone (FMNH*,FADH*) towards the red, anionic species is observed upon increasing the pH from 6.5 to 8.5. Spectrophotometric analysis of events following the mixing of oxidized CPR and NADPH (1 to 1) in a stopped-flow instrument demonstrates that the establishment of this thermodynamic equilibrium becomes a very slow process at elevated pH, indicative of a pH-gating mechanism. The final level of blue di-semiquinone formation is found to be pH independent. Stopped-flow experiments using excess NADPH over CPR provide evidence that both pH and solvent significantly influence the kinetic exposure of the blue di-semiquinone intermediate, yet the observed rate constants are essentially pH independent. Thus, the kinetic pH-gating mechanism under stoichiometric conditions is of no significant kinetic relevance for four-electron reduction, but rather modulates the observed semiquinone absorbance at 600 nm in a pH-dependent manner. The use of proton inventory experiments and primary kinetic isotope effects are described as kinetic tools to disentangle the intricate pH-dependent kinetic mechanism in CPR. Our analysis of the pH and isotope dependence in human CPR reveals previously hidden complexity in the mechanism of electron transfer in this complex flavoprotein.