NMR structures of apo L. casei dihydrofolate reductase and its complexes with trimethoprim and NADPH: contributions to positive cooperative binding from ligand-induced refolding, conformational changes, and interligand hydrophobic interactions

In order to examine the origins of the large positive cooperativity (ΔG(0)(coop) = -2.9 kcal mol(-1)) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of L. casei apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A-B loop region and part of helix B (residues 15-31) which could not be directly detected for L. casei apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to -0.95 kcals mol(-1) if 80% of A-B loop requires refolding). Comparisons of Cα-Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to -1.4 kcal mol(-1)).     

Primary isolates of human immunodeficiency virus type 1 are usually dominated by the major variants found in blood

The isolation of primary strains of human immunodeficiency virus (HIV) is an invaluable tool for assessing properties of viruses replicating in HIV-infected subjects. A common method for obtaining a primary isolate is coculture of peripheral blood mononuclear cells (PBMCs) from HIV-infected subjects with PBMCs from uninfected donors. However, such in vitro expansion may disturb the composition (identities and relative proportions of constituting viral species) of the original viral population. We developed a GeneScan assay to monitor HIV populations by detecting variants that differ in the length of the V1/V2 coding region of the envelope gene. This assay was used to compare proviral DNAs from the PBMCs of eight subjects to the corresponding primary isolates. Major variants found in uncultured PBMCs usually persisted during culturing, while the minor variants frequently disappeared, resulting in a reduction in viral diversity. The outgrowth of the initial (2 to 4 days) viral population appeared to be determined by random events. However, subsequent changes in the population were deterministic, and as a result, the compositions of primary isolates from parallel cultures were often very similar. For two of three subjects studied, the source of HIV-negative PBMCs had little effect on the composition of primary isolates, while for the third subject donor-dependent effects were observed. Overall, our results show that most primary isolates accurately represent the major viruses found in a subject's blood and that rapid population-based genotyping methods are useful for detecting isolates with perturbed viral populations.     

Analysis of resonance energy transfer in model membranes: role of orientational effects

The model of resonance energy transfer (RET) in membrane systems containing donors randomly distributed over two parallel planes separated by fixed distance and acceptors confined to a single plane is presented. Factors determining energy transfer rate are considered with special attention being given to the contribution from orientational heterogeneity of the donor emission and acceptor absorption transition dipoles. Analysis of simulated data suggests that RET in membranes, as compared to intramolecular energy transfer, is substantially less sensitive to the degree of reorientational freedom of chromophores due to averaging over multiple donor-acceptor pairs. The uncertainties in the distance estimation resulting from the unknown mutual orientation of the donor and acceptor are analyzed.     

Left and right in the amphibian world: which way to develop and where to turn?

The last decade has seen a dramatic increase in studies on the development, function and evolution of asymmetries in vertebrates, including amphibians. Here we discuss current knowledge of behavioral and anatomical asymmetries in amphibians. Behavioral laterality in the response of both adult and larval anurans to presumed predators and competitors is strong and may be related, respectively, to laterality in the telencephalon of adults and the Mauthner neurons of tadpoles. These behavior lateralities, however, do not seem to correlate with visceral asymmetries in the same animals. We briefly compare what is known about the evolution and development of asymmetry in the structure and function of amphibians with what is known about asymmetries in other chordate and non-chordate groups. Available data suggest that the majority of asymmetries in amphibians fall into two independent groups: (1) related to situs viscerum and (2) of a neurobehavioral nature. We find little evidence linking these two groups, which implies different developmental regulatory pathways and independent evolutionary histories for visceral and telencephalic lateralizations. Studies of animals other than standard model species are essential to test hypotheses about the evolution of laterality in amphibians and other chordates.     

HIV-1 Tat protein induces aberrant activation of AICDA in human B-lymphocytes from peripheral blood

Individuals infected with human immunodeficiency virus (HIV) are at increased risk for Burkitt lymphoma, a B-cell malignancy which occurs after a chromosomal translocation rearranging the MYC oncogene with an immunoglobulin gene locus, usually the IGH heavy chain gene locus. We have previously reported that the HIV protein Tat which circulates in all HIV-positive individuals whatever their immune status caused an increased rate of colocalization between IGH and MYC in B-cells nuclei. We here present in vitro evidence that Tat activates the expression of the AICDA gene that encodes the activation-induced cytidine deaminase whose physiological function is to create double-strand breaks for immunoglobulin gene maturation. In the presence of Tat, DNA damage was observed concomitantly in both MYC and IGH, followed by DNA repair by nonhomologous end joining. AICDA was further found overexpressed in vivo in peripheral blood B-cells from HIV-infected individuals. Thus, the capacity of Tat to spontaneously penetrate B-cells could be sufficient to favor the occurrence of MYC-IGH oncogenic rearrangements during erroneous repair, a plausible cause for the increased incidence of Burkitt lymphoma in the HIV-infected population.     

Maternal effects, conspecific chemical cues, and switching from parthenogenesis to gametogenesis in the cladoceran Moina macrocopa

The change in reproductive mode from parthenogenesis to gametogenesis in Cladocera is controlled by multiple environmental cues. Maternal effects are involved in the control of reproductive switching. In this study, we estimated the readiness of Moina macrocopa females to change reproduction mode under the effect of conspecific chemicals on maternal and offspring generations. The results demonstrated that information about the chemical environment was not transmitted between generations (none of the females produced diapausing eggs in the control medium irrespective of their mothers' environment). Differences in maternal energetic investments were not significant, hence the maternal environment did not affect the fecundity. However, tested animals adapted to the effect of the diapause inducing factor. When offspring of mothers cultured in crowded water were also cultured in crowded water (the constant effect of conspecific chemicals), they switched less readily to gametogenesis than offsprings of mothers cultured in fresh medium.