Structure-function relationship in a variant hemoglobin: a combined computational-experimental approach

Our study examines the functional and structural effects of amino acid substitution in the distal side of beta-chains of human Hb Duarte (alpha(2)beta(2)(62Ala-->Pro)). We have compared the functional properties of the purified Hb Duarte with those of HbA, and through proton NMR and molecular dynamics simulations we have investigated their tertiary and quaternary structures. The variant exhibits an increased oxygen affinity with a normal Hill coefficient and Bohr effect. The abnormal function of Hb Duarte is attributed to the presence of a proline residue at the beta62 position, since the functional properties of another Hb variant in the same position, Hb J-Europa (beta(62Ala-->Asp)), have been described as normal. Thereafter (1)H-NMR studies have shown that the beta62 Ala-->Pro substitution causes structural modifications of the tertiary structure of the beta globins, leaving the quaternary structure unaltered. These results have been confirmed by extensive all-atom molecular dynamics simulations. All these findings lead to the conclusion that the beta62 Ala-->Pro substitution produces a destabilization of the E-helix extending downward to the CD corner. Particularly, a cavity near the distal histidine of the beta-chains, connecting the heme pocket to the solvent, is affected, altering the functional properties of the protein molecule.     

Defining residues involved in human rhinovirus 2A proteinase substrate recognition

The 2A proteinase (2A(pro)) of human rhinoviruses (HRVs) initiates proteolytic processing by cleaving between the C-terminus of VP1 and its own N-terminus. It subsequently cleaves the host protein eIF4GI. HRV2 and HRV14 2A(pro) cleave at IITTA *GPSD and DIKSY *GLGP on their respective polyproteins. The HRV2 2A(pro) cleavage site on eIF4GI is TLSTR *GPPR. We show that HRV2 2A(pro) can self-process at the eIF4GI cleavage sequence whereas HRV14 2A(pro) cannot, due to the presence of the arginine residue at P1. The mutations A104C or A104S in HRV14 2A(pro) restored cleavage when arginine was present at P1, although not to wild-type levels. These experiments define residues which determine substrate recognition in rhinoviral 2A(pro).     

Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.     

Highly structured Asian Drosophila melanogaster populations: a new tool for hitchhiking mapping?

Mark-recapture experiments showed that D. melanogaster has high dispersal capabilities. Consistent with a highly migratory species, only very low levels of differentiation were described for D. melanogaster populations from the same continent. We reinvestigated the population structure in D. melanogaster using 49 polymorphic markers in 23 natural populations. While European and American D. melanogaster populations showed very low differentiation, Asian D. melanogaster populations were highly structured. Despite the high differentiation of Asian flies, we confirm that all non-African populations are derived from a single colonization event. We propose that the availability of D. melanogaster populations with high and low population structure provides a novel tool for the identification of ecologically important adaptations by hitchhiking mapping.     

Symmetry breaking in interspecific Drosophila hybrids is not due to developmental noise

Hybrids from crosses of different species have been reported to display decreased developmental stability when compared to their pure species, which is conventionally attributed to a breakdown of coadapted gene complexes. Drosophila subobscura and its close relative D. madeirensis were hybridized in the laboratory to test the hypothesis that genuine fluctuating asymmetry, measured as the within-individual variance between right and left wings that results from random perturbations in development, would significantly increase after interspecific hybridization. When sires of D. subobscura were mated to heterospecific females following a hybrid half-sib breeding design, F1 hybrid females showed a large bilateral asymmetry with a substantial proportion of individuals having an asymmetric index larger than 5% of total wing size. Such an anomaly, however, cannot be plainly explained by an increase of developmental instability in hybrids but is the result of some aberrant developmental processes. Our findings suggest that interspecific hybrids are as able as their parents to buffer developmental noise, notwithstanding the fact that their proper bilateral development can be harshly compromised. Together with the low correspondence between the co-variation structures of the interindividual genetic components and the within-individual ones from a Procrustes analysis, our data also suggest that the underlying processes that control (genetic) canalization and developmental stability do not share a common mechanism. We argue that the conventional account of decreased developmental stability in interspecific hybrids needs to be reappraised.     

Very low additive genetic variance and evolutionary potential in multiple populations of two rainforest Drosophila species

Most quantitative traits are thought to exhibit high levels of genetic variance and evolutionary potential. However, this conclusion may be biased by a lack of studies on nonmodel organisms and may not generalize to restricted species. A recent study on a single, southern population of the rainforest-restricted Drosophila birchii failed to find significant additive genetic variance for the desiccation resistance trait; however, it is unclear whether this pattern extends to other D. birchii populations or to other rainforest species. Here we use an animal model design to show very low levels of additive genetic variance for desiccation resistance in multiple populations of two highly sensitive rainforest species of Drosophila from tropical northeastern Australia. In contrast, relatively high levels of genetic variance were found for morphological traits in all populations of the species tested. This indicates limited evolutionary potential for evolving increased desiccation resistance in these rainforest restricted species.     

Islet-specific glucose-6-phosphatase catalytic subunit-related protein-reactive CD4+ T cells in human subjects

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is recognized as a major autoantigen for autoimmune type 1 diabetes (T1D) in the NOD mouse model. This study was undertaken to examine CD4+ T cell responses toward IGRP in human subjects. The tetramer-guided epitope mapping approach was used to identify IGRP-specific CD4+ T cell epitopes. IGRP(23-35) and IGRP(247-259) were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP(13-25) and IGRP(226-238) were identified as DRA1*0101/DRB1*0301-restricted epitopes. IGRP-specific tetramers were used to evaluate the prevalence of IGRP-reactive T cells in healthy and T1D subjects. More than 80% of subjects with either DRB1*0401 or DRB1*0301 haplotype have IGRP-specific CD4+ T cell responses for at least one IGRP epitope. IGRP-specific T cells from both healthy and T1D groups produce both gamma-IFN and IL-10. DRA1*0101/DRB1*0401 IGRP(247-259)-restricted T cells also show cross-reactivity to an epitope derived from liver/kidney glucose-6-phosphatase. The detection of IGRP-reactive T cells in both type 1 diabetic subjects and healthy subjects and recent reports of other autoreactive T cells detected in healthy subjects underscore the prevalence of potentially autoreactive T cells in the peripheral immune system of the general population.     

Absence of clinal variation in virgin retention capacity in Australian Drosophila melanogaster

The ability of virgin Drosophila melanogaster adults to retain eggs is thought to be an adaptation to persisting in temperate areas, based on differences in this trait between European and African populations, and based on seasonal changes in this trait in France. By retaining eggs in the absence of males and under conditions of poorer nutrition (conditions common in temperate areas during colder months), females reduce the wastage of resources and increase their probability of surviving spring into summer, enabling them to initiate summer population expansions. To test for variation in virgin egg retention along a climatic gradient, we characterized clinal variation in strains collected from eastern Australia extending from temperate Tasmania to tropical northern Queensland. Despite testing a large number of strains and repeated testing of the cline ends, we did not detect any evidence for clinal variation in virgin egg retention. Therefore although D. melanogaster in temperate Australia overwinter at the adult stage, there is no evidence for selection on virgin retention capacity producing clinal patterns. This contrasts with other evidence for clinal variation in egg production patterns over winter.