Antioxidant function of cytosolic sources of NADPH in yeast

The relative antioxidant functions of thiol-dependent mechanisms and of direct catalytic inactivation of H2O2 were examined using a collection of yeast mutants containing disruptions in single or multiple genes encoding two major enzymatic sources of NADPH [glucose-6-phosphate dehydrogenase (ZWF1) and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2)] and in genes encoding two major cellular peroxidases [mitochondrial cytochrome c peroxidase (CCP1) and cytosolic catalase (CTT1)]. Both types of mechanisms were found to be important for growth in the presence of exogenous H2O2. In the absence of exogenous oxidants, however, loss of ZWF1 and IDP2, but not loss of CTT1 and CCP1, was found to be detrimental not only to growth but also to viability of cells shifted to rich medium containing oleate or acetate. The loss in viability correlates with increased levels of intracellular oxidants apparently produced during normal metabolism of these carbon sources. Acute effects in DeltaZWF1DeltaIDP2 mutants following shifts to these nonpermissive media include an increase in the number of cells demonstrating a transient decrease in growth rate and in cells containing apparent nuclear DNA strand breaks. Cumulative effects are reflected in phenotypes, including sensitivity to acetate medium and a reduction in mating efficiency, that become more pronounced with time following disruption of the ZWF1 and IDP2 genes. These results suggest that cellular mechanisms dependent on NADPH are crucial metabolic antioxidants.     

Who is eating fructose within the Aedes albopictus gut microbiota?

The Asian tiger mosquito Aedes albopictus is a major public health concern because of its invasive success and its ability to transmit pathogens. Given the low availability of treatments against mosquito-borne diseases, vector control remains the most suitable strategy. The methods used thus far are becoming less effective, but recent strategies have emerged from the study of mosquito-associated microorganisms. Although the role of the microbiota in insect biology does not require further proof, much remains to be deciphered in mosquitoes, especially the contribution of the microbiota to host nutrient metabolism. Mosquitoes feed on plant nectar, composed of mostly fructose. We used stable isotope probing to identify bacteria and fungi assimilating fructose within the gut of Ae. albopictus. Mosquitoes were fed a ¹³C-labelled fructose solution for 24 h. Differences in the active microbial community according to the sex of mosquitoes were highlighted. The bacterium Lelliottia and the fungi Cladosporium and Aspergillus dominated the active microbiota in males, whereas the bacterium Ampullimonas and the yeast Cyberlindnera were the most active in females. This study is the first to investigate trophic interactions between Ae. albopictus and its microbiota, thus underscoring the importance of the microbial component in nectar feeding in mosquitoes.     

Kinetic studies of the refolding of yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.     

The effects of foraging demand on social interactions in a laboratory group of Bonnet Macaques

The relationship between foraging demand and social behavior was experimentally studied in a laboratory group of bonnet macaques. Fourteen adult animals were housed in a large outdoor enclosure containing three shallow gravelfilled circular containers that served as the foraging sites. During the experimental foraging sessions raisins were placed in the containers and the social and foraging behaviors of the group were observed for 50 min following the distribution of raisins. Three types of foraging conditions were inter-spersed with one another on different test days: (1) surface load— raisins placed on top of the gravel; (2) buried load— raisins hidden underneath the gravel; and (3) sham load— no raisins placed at the foraging sites. Three basic foraging patterns, defined along a temporal dimension, were seen. One group of animals completed 50% of their total foraging by the end of the first 15 min. A second group foraged more steadily through the session. A third group foraged late, completing 50% of their foraging during the last half of the session. The foraging patterns were similar in the buried and surface condition, although the patterns were more compressed during the surface condition. More aggression and more avoidance of other animals occurred in the buried condition than in the surface condition. Very little foraging occurred during the sham condition. There was no clear relationship between the patterns of interaction during foraging and nonforaging observation sessions. The results suggest the value of manipulative laboratory studies in examining the relationship between ecological variables and social behavior in nonhuman primates.     

Crystal structure of the yeast Phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 A crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the beta-sheet and alpha-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.     

ELECTROSHOCK-INDUCED SEIZURES AND THE TURNOVER OF BRAIN PROTEIN IN THE RAT

Abstract— A total of ten electroshock seizures (two seizures per day) were induced in rats beginning 3 days after an injection of [U-¹⁴C]glucose. Despite the intense stimulation, the labelling of the protein and nucleic acid fractions in the brains of convulsed animals decreased only slightly and not significantly. During the first 2 days after administration of [¹⁴C]glucose to untreated animals, there was a slight decrease in the specific activity of protein-bound glutamic acid relative to that of aspartic acid and the total protein fraction, suggesting the presence of a protein with a high content of glutamic acid and a rapid turnover.     

3 beta, 17 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. Kinetic evidence for the bifunctional activity at a common catalytic site

3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.     

Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10⁹ independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with K_d values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.