Functional annotation by identification of local surface similarities: a novel tool for structural genomics

Background  

Protein function is often dependent on subsets of solvent-exposed residues that may exist in a similar three-dimensional configuration in non homologous proteins thus having different order and/or spacing in the sequence. Hence, functional annotation by means of sequence or fold similarity is not adequate for such cases.     

Tracking human migrations by the analysis of the distribution of HLA alleles, lineages and haplotypes in closed and open populations

The human leucocyte antigen (HLA) system shows extensive variation in the number and function of loci and the number of alleles present at any one locus. Allele distribution has been analysed in many populations through the course of several decades, and the implementation of molecular typing has significantly increased the level of diversity revealing that many serotypes have multiple functional variants. While the degree of diversity in many populations is equivalent and may result from functional polymorphism(s) in peptide presentation, homogeneous and heterogeneous populations present contrasting numbers of alleles and lineages at the loci with high-density expression products. In spite of these differences, the homozygosity levels are comparable in almost all of them. The balanced distribution of HLA alleles is consistent with overdominant selection. The genetic distances between outbred populations correlate with their geographical locations; the formal genetic distance measurements are larger than expected between inbred populations in the same region. The latter present many unique alleles grouped in a few lineages consistent with limited founder polymorphism in which any novel allele may have been positively selected to enlarge the communal peptide-binding repertoire of a given population. On the other hand, it has been observed that some alleles are found in multiple populations with distinctive haplotypic associations suggesting that convergent evolution events may have taken place as well. It appears that the HLA system has been under strong selection, probably owing to its fundamental role in varying immune responses. Therefore, allelic diversity in HLA should be analysed in conjunction with other genetic markers to accurately track the migrations of modern humans.     

Optimal whole-body vibration settings for muscle strength and power enhancement in human knee extensors

This study compared the effects of 6-week whole-body vibration (WBV) training programs with different frequency and peak-to-peak displacement settings on knee extensor muscle strength and power. The underlying mechanisms of the expected gains were also investigated. Thirty-two physically active male subjects were randomly assigned to a high-frequency/high peak-to-peak displacement group (HH; n = 12), a low-frequency/low peak-to-peak displacement group (LL; n = 10) or a sham training group (SHAM; n = 10). Maximal voluntary isometric, concentric and eccentric torque of the knee extensors, maximal voluntary isometric torque of the knee flexors, jump performance, voluntary muscle activation, and contractile properties of the knee extensors were assessed before and after the training period. Significant improvement in knee extensor eccentric voluntary torque (P < 0.01), knee flexor isometric voluntary torque (P < 0.05), and jump performance (P < 0.05) was observed only for HH group. Regardless of the group, knee extensor muscle contractile properties (P < 0.05) were enhanced. No modification was observed for voluntary muscle activation or electrical activity of agonist and antagonist muscles. We concluded that high-frequency/high peak-to-peak displacement was the most effective vibration setting to enhance knee extensor muscle strength and jump performance during a 6-week WBV training program and that these improvements were not mediated by central neural adaptations.     

Single-cell responses to ionizing radiation

While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of “housekeeping” genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories—those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.     

Methotrexate enhances 3T3-L1 adipocytes hypertrophy

Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and ³H-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 μM). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. ³H-Deoxyglucose (³H-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and ³H-thymidine incorporation (P?<?0.05). MTX (0.1 μM) treatment increased lipid accumulation and basal ³H-DG uptake by adipocytes (P?<?0.05). In 0.1 μM MTX-treated adipocytes, insulin stimulation did not result in an increase of ³H-DG uptake, contrarily to what was observed in control cells. These results demonstrate that methotrexate interferes with adipocyte proliferation and promotes the hypertrophic growth of adipocytes. These molecular effects may have implications on metabolic profile of RA patients treated with MTX.     

Simultaneous production of triploid and haploid eggs by triploid Squalius alburnoides (Teleostei: Cyprinidae)

The hybrid minnow Squalius alburnoides comprises diploid and polyploid forms with altered modes of reproduction. In the present paper, we report a cross where a triploid female generated both large, triploid and small, haploid eggs simultaneously, which were fertilized with S. pyrenaicus sperm. Although the large eggs were rarer (15%), they originated offspring with higher survivorship, so that tetraploids were dominant among the surviving siblings. The cross yielded apparently all female progeny. Inheritance patterns were inferred using four microsatellite markers and NORs (Nucleolus Organizer Regions) phenotypes, and suggested that haploid eggs were probably produced by an atypical hybridogenesis, in which the elimination of the unmatched genome permitted random segregation and recombination between the homospecific genomes, while the triploid eggs were clonal. The present results suggest that the occurrence of triploid unreduced eggs may be a new route for the natural tetraploidization in the complex.     

Identification of the human rhinovirus serotype 1A binding site on the murine low-density lipoprotein receptor by using human-mouse receptor chimeras

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.