Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD⁺ and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD⁺ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k _cat/K _m) with NADP⁺. The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00?±?0.13 U?mg^?1 and a K _{m, NADP} ⁺ of 0.147?±?0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP⁺-accepting FDH (v _max, 10.25?±?1.63 U?mg^?1). However, the half-saturation constant for NADP⁺ (K _{m, NADP} ⁺ , 0.92?±?0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.     

Similar Performance of Diploid and Haploid Males in an Ant Species without Inbreeding Avoidance

Under haplodiploidy, a characteristic trait of all Hymenoptera, females develop from fertilised eggs, and males from unfertilised ones. Males are therefore typically haploid. Yet, inbreeding can lead to the production of diploid males that often fail in development, are sterile or are of lower fertility. In most Hymenoptera, inbreeding is avoided by dispersal flights of one or both sexes, leading to low diploid male loads. We investigated causes for the production of diploid males and their performance in a highly inbred social Hymenopteran species. In the ant Hypoponera opacior, inbreeding occurs between wingless sexuals, which mate within the mother nest, whereas winged sexuals outbreed during mating flights earlier in the season. Wingless males mate with queen pupae and guard their mating partners. We found that they mated randomly with respect to relatedness, indicating that males do not avoid mating with close kin. These frequent sib‐matings lead to the production of diploid males, which are able to sire sterile triploid offspring. We compared mating activity and lifespan of haploid and diploid wingless males. As sexual selection acts on the time of emergence and body size in this species, we also investigated these traits. Diploid males resembled haploid ones in all investigated traits. Hence, albeit diploid males cannot produce fertile offspring, they keep up with haploid males in their lifetime mating success. Moreover, by fathering viable triploid workers, they contribute to the colonies' work force. In conclusion, the lack of inbreeding avoidance led to frequent sib‐matings of wingless sexuals, which in turn resulted in the regular production of diploid males. However, in contrast to many other Hymenopteran species, diploid males exhibit normal sexual behaviour and sire viable, albeit sterile daughters.     

Serum-Dependent Enhancement of Coxsackievirus B4-Induced Production of IFNα, IL-6 and TNFα by Peripheral Blood Mononuclear Cells

Only a few reports have been published on the interactions between Coxsackievirus B4 (CVB4) and human peripheral blood mononuclear cells (PBMC) but have not been extensively documented. Human serum containing non-neutralizing anti-CVB4 antibodies increased CVB4-induced synthesis of IFNα by PBMC. In this study, we determined if CVB4 and human serum have the ability to activate inflammatory cytokines in addition to IFNα in PBMC cultures. PBMC from healthy donors were inoculated with infectious, inactivated CVB4 or with CVB4 incubated with dilutions of human serum or polyvalent IgG with anti-CVB4 activity. Levels of IFNα, TNFα, IL-6, IL-12, IFNγ and IL-10 in the cell-free supernatants of PBMC cultures were measured using ELISA. Infection was assessed by real-time PCR. PBMC inoculated with CVB4 produced inflammatory cytokines but not IFNα. When CVB4 was incubated with serum or IgG, IFNα was detected in the culture supernatants, and high concentrations of TNFα and IL-6 were measured. The concentrations of TNFα and IL-6 were not reduced in cultures inoculated with inactivated CVB4, whereas the IgG-dependent enhancement of IFNα, IL-6 and TNFα production with inactivated virus was suppressed. The potentiation of IFNα production was associated with a high intracellular viral load. Infectious and non-infectious CVB4 can induce the production of inflammatory cytokines but not IFNα by PBMC. High levels of IFNα, in addition to TNFα and IL-6, in culture supernatants were obtained when infectious CVB4 was combined with immune serum or IgG, and they were associated with high amounts of intracellular viral RNA.     

Peptide-binding motifs associated with MHC molecules common in Chinese rhesus macaques are analogous to those of human HLA supertypes and include HLA-B27-like alleles

Chinese rhesus macaques are of particular interest in simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) research as these animals have prolonged kinetics of disease progression to acquired immunodeficiency syndrome (AIDS), compared to their Indian counterparts, suggesting that they may be a better model for HIV. Nevertheless, the specific mechanism(s) accounting for these kinetics remains unclear. The study of major histocompatibility complex (MHC) molecules, including their MHC/peptide-binding motifs, provides valuable information for measuring cellular immune responses and deciphering outcomes of infection and vaccine efficacy. In this study, we have provided detailed characterization of six prevalent Chinese rhesus macaque MHC class I alleles, yielding a combined phenotypic frequency of 29 %. The peptide-binding specificity of two of these alleles, Mamu-A2*01:02 and Mamu-B*010:01, as well as the previously characterized allele Mamu-B*003:01 (and Indian rhesus Mamu-B*003:01), was found to be analogous to that of alleles in the HLA-B27 supertype family. Specific alleles in the HLA-B27 supertype family, including HLA-B*27:05, have been associated with long-term nonprogression to AIDS in humans. All six alleles characterized in the present study were found to have specificities analogous to HLA supertype alleles. These data contribute to the concept that Chinese rhesus macaque MHC immunogenetics is more similar to HLA than their Indian rhesus macaque counterparts and thereby warrants further studies to decipher the role of these alleles in the context of SIV infection.     

Mitochondrial dynamics generate equal distribution but patchwork localization of respiratory Complex I

Highly dynamic mitochondrial morphology is a prerequisite for fusion and fission. Mitochondrial fusion may represent a rescue mechanism for impaired mitochondria by exchanging constituents (proteins, lipids and mitochondrial DNA) and thus maintaining functionality. Here we followed for the first time the dynamics of a protein complex of the respiratory chain during fusion and fission. HeLa cells with differently labelled respiratory Complex I were fused and the dynamics of Complex I were investigated. The mitochondrial proteins spread throughout the whole mitochondrial population within 3 to 6 h after induction of cell fusion. Mitochondria of fused cells displayed a patchy substructure where the differently labelled proteins occupied separate and distinct spaces. This patchy appearance was already – although less pronounced – observed within single mitochondria before fusion, indicating a specific localization of Complex I with restricted diffusion within the inner membrane. These findings substantiate the view of a homogenous mitochondrial population due to constantly rearranging mitochondria, but also indicate the existence of distinct inner mitochondrial sub-compartments for respiratory chain complexes.     

Relative Risk of Visceral Leishmaniasis in Brazil: A Spatial Analysis in Urban Area

Background

Visceral leishmaniasis (VL) is a vector-borne disease whose factors involved in transmission are poorly understood, especially in more urban and densely populated counties. In Brazil, the VL urbanization is a challenge for the control program. The goals were to identify the greater risk areas for human VL and the risk factors involved in transmission.

Methodology

This is an ecological study on the relative risk of human VL. Spatial units of analysis were the coverage areas of the Basic Health Units (146 small-areas) of Belo Horizonte, Minas Gerais State, Brazil. Human VL cases, from 2007 to 2009 (n = 412), were obtained in the Brazilian Reportable Disease Information System. Bayesian approach was used to model the relative risk of VL including potential risk factors involved in transmission (canine infection, socioeconomic and environmental features) and to identify the small-areas of greater risk to human VL.

Principal Findings

The relative risk of VL was shown to be correlated with income, education, and the number of infected dogs per inhabitants. The estimates of relative risk of VL were higher than 1.0 in 54% of the areas (79/146). The spatial modeling highlighted 14 areas with the highest relative risk of VL and 12 of them are concentrated in the northern region of the city.

Conclusions

The spatial analysis used in this study is useful for the identification of small-areas according to risk of human VL and presents operational applicability in control and surveillance program in an urban environment with an unequal spatial distribution of the disease. Thus the frequent monitoring of relative risk of human VL in small-areas is important to direct and prioritize the actions of the control program in urban environment, especially in big cities.     

Tracking of human Y receptors in living cells--a fluorescence approach

Non-invasive methods for studying biological processes in living cells have become very important, also in the field of GPCR biochemistry. Great advancements in the application of fluorescence techniques as well as in the development and improvement of novel fluorophores allow the visualization of dynamic processes. Using these technologies, problems concerning receptor biosynthesis, internalization, recycling and degradation can be investigated. Here we compare the application of the different fluorescent tags EYFP, Lumiotrade mark and SNAPtrade mark to track hY(1) and hY(5) receptors in living cells.     

New Insights into the Structure and Mode of Action of Mo-CBP3, an Antifungal Chitin-Binding Protein of Moringa oleifera Seeds

Mo-CBP₃ is a chitin-binding protein purified from Moringa oleifera Lam. seeds that displays inhibitory activity against phytopathogenic fungi. This study investigated the structural properties and the antifungal mode of action of this protein. To this end, circular dichroism spectroscopy, antifungal assays, measurements of the production of reactive oxygen species and microscopic analyses were utilized. Mo-CBP₃ is composed of 30.3% α-helices, 16.3% β-sheets, 22.3% turns and 30.4% unordered forms. The Mo-CBP₃ structure is highly stable and retains its antifungal activity regardless of temperature and pH. Fusarium solani was used as a model organism for studying the mechanisms by which this protein acts as an antifungal agent. Mo-CBP₃ significantly inhibited spore germination and mycelial growth at 0.05 mg.mL⁻¹. Mo-CBP₃ has both fungistatic and fungicidal effects, depending on the concentration used. Binding of Mo-CBP₃ to the fungal cell surface is achieved, at least in part, via electrostatic interactions, as salt was able to reduce its inhibitory effect. Mo-CBP₃ induced the production of ROS and caused disorganization of both the cytoplasm and the plasma membrane in F. solani cells. Based on its high stability and specific toxicity, with broad-spectrum efficacy against important phytopathogenic fungi at low inhibitory concentrations but not to human cells, Mo-CBP₃ has great potential for the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogens.