NMR investigations of protein-carbohydrate interactions: refined three- dimensional structure of the complex between hevein and methyl beta- chitobioside

The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from1H-NMR titration experiments. The association constants increase in the order GlcNAc-alpha(1-->6)-Man < GlcNAc < benzyl-beta-GlcNAc < p-nitrophenyl-beta-GlcNAc < chitobiose < p- nitrophenyl-beta-chitobioside < methyl-beta-chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from van't Hoff analysis. The driving force for the binding process is provided by a negative DeltaH0which is partially compensated by negative DeltaS0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-proton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this protein- carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2.      

Diversity among bacteria isolated from the deep subsurface

Culturable bacteria from the deep subsurface (179 m) at Cerro Negro, New Mexico were isolated and characterized. The average number of viable aerobic bacteria was estimated to be 5×10⁵g^–1 of sediment, but only about 0.1% of these could be recovered on agar medium when incubated under aerobic conditions. Of 158 strains isolated from this depth, 92 were characterized by cellular fatty acid profiles (FAME), 36 by analysis of partial 16S rDNA sequences, and 44 by rep-PCR genome fingerprint analysis using three different sets of oligonucleotide primers (REP, BOX, or ERIC). These analyses showed the majority of isolates (67%) were Gram-positive bacteria and primarily members of genera with a high %G+C DNA. The remaining isolates were -subdivisionProteobacteria (19%) and members of the flavobacteria group (14%). The diversity indices based on these different methods of characterization were very high suggesting this subsurface habitat harbors a highly diverse microbial community.     

Phylogenetic reconstruction of the Drosophila obscura group, on the basis of mitochondrial DNA

We have constructed restriction-site maps of the mtDNAs in 13 species and one subspecies of the Drosophila obscura group. The traditional division of this group into two subgroups (affinis and obscura) does not correspond to the phylogeny of the group, which shows two well- defined clusters (the Nearctic affinis and pseudoobscura subgroups) plus a very heterogeneous set of anciently diverged species (the Palearctic obscura subgroup). The mtDNA of Drosophila exhibits a tendency to evolve toward high A+T values. This leads to a "saturation" effect that (1) begets an apparent decrease in the rate of evolution as the time since the divergence of taxa increases and (2) reduces the value that mtDNA restriction analysis has for the phylogenetic reconstruction of Drosophila species that are not closely related.      

Intracellular calcium buffering capacity in isolated squid axons

Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 μmol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/μM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 μM is taken up by this system.     

Retention of plastic-tipped dart tags in African tigerfish Hydrocynus vittatus

Estimates of tag retention and tagging-related mortality are essential for mark-recapture experiments. Mortality and tag loss were estimated from 15 tigerfish Hydrocynus vittatus marked using Hallmark model PDL plastic-tipped dart tags released into a 1 730 m² pond at Kamutjonga Inland Fisheries Institute, Namibia, and inspected bi-monthly for the presence or absence of tags. No mortality was observed during the experiment. All marked fish had lost their tags after 10 months and 50% tag loss was estimated at 3.9 months. The high tag loss rate indicates that PDL plastic-tipped dart tags are not suitable for long-term studies on this species.     

Maximum in the Middle: Nonlinear Response of Microbial Plankton to Ultraviolet Radiation and Phosphorus

The responses of heterotrophic microbial food webs (HMFW) to the joint action of abiotic stressors related to global change have been studied in an oligotrophic high-mountain lake. A 2×5 factorial design field experiment performed with large mesocosms for >2 months was used to quantify the dynamics of the entire HMFW (bacteria, heterotrophic nanoflagellates, ciliates, and viruses) after an experimental P-enrichment gradient which approximated or surpassed current atmospheric P pulses in the presence vs. absence of ultraviolet radiation. HMFW underwent a mid-term (<20 days) acute development following a noticeable unimodal response to P enrichment, which peaked at intermediate P-enrichment levels and, unexpectedly, was more accentuated under ultraviolet radiation. However, after depletion of dissolved inorganic P, the HMFW collapsed and was outcompeted by a low-diversity autotrophic compartment, which constrained the development of HMFW and caused a significant loss of functional biodiversity. The dynamics and relationships among variables, and the response patterns found, suggest the importance of biotic interactions (predation/parasitism and competition) in restricting HMFW development, in contrast to the role of abiotic factors as main drivers of autotrophic compartment. The response of HMFW may contribute to ecosystem resilience by favoring the maintenance of the peculiar paths of energy and nutrient-mobilization in these pristine ecosystems, which are vulnerable to threats by the joint action of abiotic stressors related to global change.     

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

Background  

Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear.     

Molecular population genetics of the alcohol dehydrogenase locus in the Hawaiian drosophilid D. mimica

Sequence variation among 10 alleles of the alcohol dehydrogenase (Adh) gene of the Hawaiian drosophilid D. mimica was analyzed with reference to the evolutionary history of the Hawaiian subgroup as well as to levels and patterns of polymorphism of the Adh gene in continental drosophilid species. The Adh gene of D. mimica is less polymorphic than that of other drosophilid species, and no replacement substitutions were found. Statistical analyses of the Adh alleles suggested the action of balancing selection and revealed significant linkage disequilibrium among three of the variable sites. The effective population size was estimated to be only slightly smaller than that of continental species and, surprisingly, on the same order of magnitude as the actual size.      

Mapping the SRY gene in Microtus cabrerae: a vole species with multiple SRY copies in males and females.

The SRY gene is a single-copy, male-specific gene, located on the Y chromosome in most mammals. However, recently we have described the presence of multiple polymorphic copies of this gene in both males and females of the vole species Microtus cabrerae. Here, we present the chromosomal localization of SRY gene copies in this species by fluorescent in situ hybridization (FISH). This technique localized these gene copies in the short arm, and hence in the euchromatic region, of the Y chromosome. Furthermore, several copies of the SRY gene are located on the X chromosome. These copies are spread along the entire heterochromatic region of the X chromosome, occupying the whole short arm, the centromeric region, and the pericentromeric region of the long arm.     

A globally occurring indel polymorphism in the promoter of the IFNA2 gene is not associated with severity of malaria but with the positivity rate of HCV

Background

Glutathione S-transferases (GSTs) is a genetic factor for many diseases and exhibits great diversities among various populations. We assessed association of the genotypes of Glutathione S-transferases Omega-1 (GSTO1) A140D with ethnicity in China.

Results

Peripheral blood samples were obtained from 1314 individuals from 14 ethnic groups. Polymorphisms of GSTO1 A140D were measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Logistic regression was employed to adjustment for regional factor. The frequency of GSTO1 140A allele was 15.49% in the total 14 ethnic populations. Compared to Han ethnic group, two ethnic populations were more likely to have AA or CA genotype [odds ratio (OR): 1.77, 95% confidence interval (95% CI): 1.05–2.98 for Uygur and OR: 1.78, 95% CI: 1.18–2.69 for Hui]. However, there were no statistically significant differences across 14 ethnic groups when region factor was adjusted. In Han ethnicity, region was significantly associated with AA or CA genotype. Han individuals who resided in North-west of China were more likely to have these genotypes than those in South of China (OR: 1.63, 95% CI: 1.21–2.20).

Conclusion

The prevalence of the GSTO1 140A varied significantly among different regional populations in China, which showed that geography played a more important role in the population differentiation for this allele than the ethnicity/race.