Increased Synapse Formation Obtained by T Cell Epitopes Containing a CxxC Motif in Flanking Residues Convert CD4+ T Cells into Cytolytic Effectors

The nature of MHC class II-binding epitopes not only determines the specificity of T cell responses, but may also alter effector cell functions. Cytolytic CD4+ T cells have been observed primarily in anti-viral responses, but very little is known about the conditions under which they can be elicited. Their potential as regulators of immune responses, however, deserves investigations. We describe here that inclusion of a thiol-disulfide oxidoreductase motif within flanking residues of class II-restricted epitopes results, both in vitro and in vivo, in elicitation of antigen-specific cytolytic CD4+ T cells through increased synapse formation. We show that both naïve and polarized CD4+ T cells, including Th17 cells, can be converted by cognate recognition of such modified epitopes. Cytolytic CD4+ T cells induce apoptosis on APCs by Fas-FasL interaction. These findings potentially open the way towards a novel form of antigen-specific immunosuppression.     

Relative orbitonasal overlap in african great apes and humans quantified by the automatic determination of horizontal and vertical lines of reference

The relative positions of the orbital and nasal openings in African apes and humans were studied by a new methodological approach based on the automatic determination, by image analysis techniques, of horizontal and vertical lines of reference. The material used consisted ofGorilla gorilla (38 males and 20 females),Pan troglodytes (19 males and 13 females), and modernHomo spaiens (51 males and 41 females). This allowed the relative positions of the orbital and nasal openings to be quantified by the determination of medio-lateral and vertical orbitonasal indices of overlap. In all the species studied, a medio-lateral orbitonasal overlap was systematically observed. This indicates that nasal breadth is always larger than interorbital distance. Medio-lateral overalp was greatest inGorilla, reduced inHomo, and intermediate inPan. By contrast, onlyHomo presents systematically a vertical overlap: a vertical overlap was sometimes observed inPan, but never inGorilla. Homo presented the greatest vertical overlap, andGorilla the least; the disposition inPan was intermediate. The interspectific study of the relationships between medio-lateral and vertical overlap inGorilla, Pan, andHomo demonstrated that an increase in veritical overlap was correlated with a decrease of medio-lateral overlap. Sexual dimorphism in orbitonasal relationships was systematically greatest inGorilla, and reduced inPan andHomo, this is also the case for the orbital, nasal, and orbitonasal parameters measured in this study. All these results provide interesting elements for understanding the morphological evolution of the middle face in hominoids.     

A disulfide bridge near the active site of carbapenem-hydrolyzing class A β-lactamases might explain their unusual substrate profile

Bacterial resistance to β-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of β-lactamases, enzymes that efficiently hydrolyze the amide bond of the β-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine β-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new β-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of β-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the “classical” TEM-1 β-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238. Proteins 27:47–58 © 1997 Wiley-Liss, Inc.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions

We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.     

Role of Mental Retardation-Associated Dystrophin-Gene Product Dp71 in Excitatory Synapse Organization,Synaptic Plasticity and Behavioral Functions

Background

Duchenne muscular dystrophy (DMD) is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR), likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood.

Methodology/Principal Findings

Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory.

Conclusions/Significance

Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.     

Estimation of demo-genetic model probabilities with Approximate Bayesian Computation using linear discriminant analysis on summary statistics

Comparison of demo‐genetic models using Approximate Bayesian Computation (ABC) is an active research field. Although large numbers of populations and models (i.e. scenarios) can be analysed with ABC using molecular data obtained from various marker types, methodological and computational issues arise when these numbers become too large. Moreover, Robert et al. (Proceedings of the National Academy of Sciences of the United States of America, 2011, 108, 15112) have shown that the conclusions drawn on ABC model comparison cannot be trusted per se and required additional simulation analyses. Monte Carlo inferential techniques to empirically evaluate confidence in scenario choice are very time‐consuming, however, when the numbers of summary statistics (Ss) and scenarios are large. We here describe a methodological innovation to process efficient ABC scenario probability computation using linear discriminant analysis (LDA) on Ss before computing logistic regression. We used simulated pseudo‐observed data sets (pods) to assess the main features of the method (precision and computation time) in comparison with traditional probability estimation using raw (i.e. not LDA transformed) Ss. We also illustrate the method on real microsatellite data sets produced to make inferences about the invasion routes of the coccinelid Harmonia axyridis. We found that scenario probabilities computed from LDA‐transformed and raw Ss were strongly correlated. Type I and II errors were similar for both methods. The faster probability computation that we observed (speed gain around a factor of 100 for LDA‐transformed Ss) substantially increases the ability of ABC practitioners to analyse large numbers of pods and hence provides a manageable way to empirically evaluate the power available to discriminate among a large set of complex scenarios.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.