Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation,Kinetics and Evolutionary Studies

Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD⁺ and NADP⁺ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP⁺ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2''-phosphate of NADP⁺, but the same residues formed no observable interactions in the case of NAD⁺. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the k_cat/K_M value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP⁺, only R50 makes a contribution (about -1 kcal/mol) to NAD⁺ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP⁺ from NAD⁺. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP⁺-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD⁺ in the case of the NADP⁺-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.     

Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37’s ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.     

Carbohydrates of the venoms of Bothrops asper of Costa Rica. Quantitative study

The venom of the Central American Bothrops asper, previously classified as B. atrox, is very rich in carbohydrates, both in the free state and forming a part of glycoproteins. It also contains neutral sugars (hexoses), methylpentoses, hexosamines and sialic acid. There is a significant difference in the quantity of carbohydrates in the venom of B. asper as compared to that of the South American B. atrox, thus further documenting the different taxonomic position of these two species.     

Lack of prion transmission by sexual or parental routes in experimentally infected hamsters

Prion diseases are a group of neurodegenerative disorders affecting humans as well as captive and wild animals. The mechanisms and routes governing the natural spread of prions are not completely understood and several hypotheses have been proposed. In this study, we analyzed the effect of gender in prion incubation period, as well as the possibility of prion transmission by sexual and parental contact using 263K infected hamsters as a model. Our results show that males have significantly longer incubation periods compared with females when exposed to the same quantity of infectious material. Importantly, no evidence of sexual or parental prion transmission was found, even 500 d after sexual contact or birth, respectively. Western blotting and PMCA were unable to detect sub-clinical levels of PrP^Sc in experimental subjects, suggesting a complete absence of prion transmission by these routes. Our results show that sexual and parental transmission of prions does not occur in this model. It remains to be studied whether this conclusion is valid also for other prion strains and species.     

Inferring the Relative Resilience of Alternative States

Ecological systems may occur in alternative states that differ in ecological structures, functions and processes. Resilience is the measure of disturbance an ecological system can absorb before changing states. However, how the intrinsic structures and processes of systems that characterize their states affects their resilience remains unclear. We analyzed time series of phytoplankton communities at three sites in a floodplain in central Spain to assess the dominant frequencies or “temporal scales” in community dynamics and compared the patterns between a wet and a dry alternative state. The identified frequencies and cross-scale structures are expected to arise from positive feedbacks that are thought to reinforce processes in alternative states of ecological systems and regulate emergent phenomena such as resilience. Our analyses show a higher species richness and diversity but lower evenness in the dry state. Time series modeling revealed a decrease in the importance of short-term variability in the communities, suggesting that community dynamics slowed down in the dry relative to the wet state. The number of temporal scales at which community dynamics manifested, and the explanatory power of time series models, was lower in the dry state. The higher diversity, reduced number of temporal scales and the lower explanatory power of time series models suggest that species dynamics tended to be more stochastic in the dry state. From a resilience perspective our results highlight a paradox: increasing species richness may not necessarily enhance resilience. The loss of cross-scale structure (i.e. the lower number of temporal scales) in community dynamics across sites suggests that resilience erodes during drought. Phytoplankton communities in the dry state are therefore likely less resilient than in the wet state. Our case study demonstrates the potential of time series modeling to assess attributes that mediate resilience. The approach is useful for assessing resilience of alternative states across ecological and other complex systems.     

Plankton biodiversity in a landscape of shallow water bodies (Mediterranean coast,Spain)

Hydrobiologia - A large spatial heterogeneity was detected in La Safor, a coastal area with different kinds of small and shallow water bodies. The area exhibits a sharp gradient in eutrophication...     

Homocysteine contribution to DNA damage in cystathionine β-synthase-deficient patients

High blood levels of homocysteine (Hcy) are found in patients affected by homocystinuria, a genetic disorder caused by deficiency of cystathionine β-synthase (CBS) activity, as well as in nutritional deficiencies (vitamin B₁₂ or folate) and in abnormal renal function. We previously demonstrated that lipid and protein oxidative damage is increased and the antioxidant defenses diminished in plasma of CBS-deficient patients, indicating that oxidative stress is involved in the pathophysiology of this disease. In the present work, we extended these investigations by evaluating DNA damage through the comet assay in peripheral leukocytes from CBS-deficient patients, as well as by analyzing of the in vitro effect of Hcy on DNA damage in white blood cells. We verified that DNA damage was significantly higher in the CBS-deficient patients under treatment based on a protein-restricted diet and pyridoxine, folic acid, betaine and vitamin B₁₂ supplementation, when compared to controls. Furthermore, the in vitro study showed a concentration-dependent effect of Hcy inducing DNA damage. Taken together, the present data indicate that DNA damage occurs in treated CBS-deficient patients, possibly due to high Hcy levels.