Dissociation of AMPK activity and ACCbeta phosphorylation in human muscle during prolonged exercise

During prolonged, low intensity exercise, the type of substrate utilized varies with time. If 5' AMP-activated protein kinase (AMPK) regulates muscle metabolism during exercise, signaling through AMPK would be expected to change in concordance with changes in substrate utilization. Six healthy, young males cycled (approximately 45% VO(2peak)) until exhaustion (approximately 3.5h). During exercise, leg glucose uptake and rate of glycogenolysis gradually decreased whereas free fatty acid uptake gradually increased. In the thigh muscle, the alpha AMPK subunits became progressively more phosphorylated on Thr(172) during exercise eliciting a parallel increase in alpha2 but not alpha1 AMPK activity. In contrast, after 1h of exercise, Ser(221) phosphorylation of acetyl-CoA carboxylase-beta (ACCbeta) peaked at 1h of exercise and returned to resting levels at exhaustion. Protein expression of alpha2 AMPK, alpha1 AMPK or ACCbeta did not change with time. These data suggest that AMPK signaling is not a key regulatory system of muscle substrate combustion during prolonged exercise and that marked activation of AMPK via phosphorylation is not sufficient to maintain an elevated ACCbeta Ser(221) phosphorylation during prolonged exercise.     

Peptides trap the human immunodeficiency virus type 1 envelope glycoprotein fusion intermediate at two sites

Human immunodeficiency virus type 1 (HIV-1) entry into target cells requires folding of two heptad-repeat regions (N-HR and C-HR) of gp41 into a trimer of N-HR and C-HR hairpins, which brings viral and target cell membranes together to facilitate membrane fusion. Peptides corresponding to the N-HR and C-HR of gp41 are potent inhibitors of HIV infection. Here we report new findings on the mechanism of inhibition of a N-HR peptide and compare these data with inhibition by a C-HR peptide. Using intact envelope glycoprotein (Env) under fusogenic conditions, we show that the N-HR peptide preferentially binds receptor-activated Env and that CD4 binding is sufficient for triggering conformational changes that allow the peptide to bind Env, results similar to those seen with the C-HR peptide. However, activation by both CD4 and chemokine receptors further enhances Env binding by both peptides. We also show that a nonconservative mutation in the N-HR of gp41 abolishes C-HR peptide but not N-HR peptide binding to gp41. These results indicate that there are two distinct sites in receptor-activated Env that are potential targets for drug or vaccine development.     

Towards a new attenuating compound: a potentiometric, spectrophotometric and NMR equilibrium study on Fe(III), Al(III) and a new tetradentate mixed bisphosphonate-hydroxypyridinonate ligand

Coordination properties toward Fe(III) and Al(III) of a mixed bisphosphonate-hydroxypyridinonate ligand are presented. Potentiometric, spectrophotometric and NMR results allowed to conclude that Fe(III) and Al(III) coordination takes place on the pyridinone moiety. The high steric hindrance prevents the possibility of simultaneous coordination of both groups to the same metal ion. Quantum mechanical calculations confirm this finding allowing to determine the minimal length of the linker necessary for a stable conformation of complexes in which Fe(III) is coordinated both by pyridinone and bisphosphonate groups.     

Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins

Much effort has been dedicated to the design of significantly red shifted variants of the green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been based on classical engineering with the 20 canonical amino acids. We report here an expansion of these efforts by incorporation of an amino substituted variant of tryptophan into the "cyan" GFP mutant, which turned it into a "gold" variant. This variant possesses a red shift in emission unprecedented for any avFP, similar to "red" FPs, but with enhanced stability and a very low aggregation tendency. An increasing number of non-natural amino acids are available for chromophore redesign (by engineering of the genetic code) and enable new general strategies to generate novel classes of tailor-made GFP proteins.     

Salts enhance both protein stability and amyloid formation of an immunoglobulin light chain

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO₄²⁻ conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.     

Genetic structure of populations of Calomys laucha (muridae, sigmodontinae) from central Argentina

The genetic structure of populations of the Sigmodontinae rodent Calomys laucha was studied by means of allozyme electrophoresis. This highly opportunistic species is found preferably in periodically perturbed habitats of crop fields in central Argentina, where it can attain very high densities. A total of 17 enzymatic proteins assayed gave information on 25 loci; only four were monomorphic in the seven populations studied. Levels of genetic variability (H_o from 0.144 to 0.171; P_95% from 44% to 56%) were higher than mean values reported for mammals and rodents. These high levels of heterozygosity could be maintained by large populations that do not experience great fluctuations in size, or by a social structure consistent in many small breeding groups that are formed and dissappear every breeding season. Genetic differentiation at a macrogeographical scale (θ=0.018) was low but statistically significant, and showed no correlation with geographic distance between pairs of populations. The pattern of population differentiation found is compatible with a relatively recent range expansion.     

Functional-cranial approach to the influence of economic strategy on skull morphology

Environmental factors are assumed to play an important role in the shaping of craniofacial morphology. Here we propose a statistical approach which can be of utility in estimating the magnitude and localization of a particular nongenetic factor upon the specific functional components of the skull. Our analysis is a combination of previous attempts of apportionment of variance and the application of craniofunctional theory. The effect of subsistence strategy on craniofacial functional components was studied on 18 populations of hunter-gatherers and farmers from South America. Results demonstrate that the environmental factors studied likely influenced the masticatory component's size and shape. Even when this effect is not large enough to clearly differentiate among subsistence strategies (since whole craniofacial variation among populations remains greater), the method used here provides interesting clues to localize plastic or adaptive responses to external stimuli.