Revisitation of the βCl-elimination reaction of D-amino acid oxidase: new interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms

D-amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl(-) from βCl-D-alanine via abstraction of the substrate α-H as H(+) ("carbanion mechanism") (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-βCl-D-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈50 s(-1). This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail.     

Histone deacetylase inhibition enhances self renewal and cardioprotection by human cord blood-derived CD34 cells

Background

Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs), have been extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains, however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of “enhancement” strategies has been therefore sought to improve repair ability of these cells and increase the clinical benefit.

Principal Findings

Pharmacologic inhibition of histone deacetylases (HDACs) is known to enhance hematopoietic stem cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in vitro. In the present study cord blood-derived CD34⁺ were pre-conditioned with the HDAC inhibitor Valproic Acid. This treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an immunodeficient mouse model of myocardial infarction.

Conclusions

Our results show that HDAC blockade leads to phenotype changes in CD34⁺ cells with enhanced self renewal and cardioprotection.     

Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

High potency improvements to weak aryl uracil HCV polymerase inhibitor leads

Described herein is the development of a potent non-nucleoside, small molecule inhibitor of genotype 1 HCV NS5B Polymerase. A 23 μM inhibitor that was active against HCV polymerase was further elaborated into a potent single-digit nanomolar inhibitor of HCV NS5B polymerase by additional manipulation of the R and R₁ substituents. Subsequent modifications to improve physical properties were made in an attempt to achieve an acceptable pharmacokinetic profile.     

Multistep enzyme catalysed deracemisation of 2-naphthyl alanine

Kinetic parameters of d-amino acid oxidase from R. gracilis (DAAO) towards d-2-naphthyl alanine (d-2-NAla) and of l-aspartate amino transferase (l-AAT) from Escherichia coli towards 2-naphthyl pyruvate (2-NPA) were measured. The two enzymes were then combined in a one-pot reaction in which DAAO was used to generate 2-NPA which was the substrate of l-AAT in the presence of cysteine sulphinic acid (CSA) as an amino donor. The combined reactions afforded enantiomerically pure l-2-NAla in almost quantitative yield. The extremely low water solubility of 2-NAla can be partially overcome by running the biotransformation in suspension with higher formal concentration. In these conditions multiple enzyme additions are required.     

l-Amino acid oxidase as biocatalyst: a dream too far?

l-Amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the “true” LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with “narrow” or “strict” substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

Prelacteal feeding and associated factors in Ethiopia: systematic review and meta-analysis

Background

Prelacteal feeding can be defined as giving any solid or liquid foods other than breast milk during the first three days after birth. It affects timely initiation of breastfeeding and exclusive breastfeeding practices. Even though the issue was investigated in Ethiopia, fragmented and inconsistent findings were reported. Therefore, the main objective of this meta-analysis was to estimate the pooled prevalence of prelacteal feeding and associated factors in Ethiopia.

Methods

The preferred reporting items for systematic reviews and meta-analyses guideline was followed. Articles were systematically searched through different searching mechanisms. Joanna Briggs Institute Meta-Analysis of Statistics Assessment and Review Instrument adapted for cross-sectional study design was used for quality assessment of each individual study. The total of 28 studies were included and analyzed. The random effect model was used to estimate the pooled prevalence; subgroup analysis and meta-regression were performed to identify the probable source of heterogeneity. Both Egger’s, and Begg’s test were used to check publication bias. The effects between associated factor variables, and prelacteal feeding practices were tested.

Results

A total of 492 studies were retrieved and 28 studies were included in the meta-analysis. The pooled prevalence of prelacteal feeding practice in Ethiopia was 25.29% (95% Confidence Interval [CI] 17.43, 33.15) with severe heterogeneity (I² =?99.7, p <?0.001) and no publication bias. Antenatal care (Odds Ratio [OR] 0.25, 95% CI 0.09, 0.69), counselling on infant feeding (OR 0.37, 95% CI 0.22, 0.63), timely initiation of breastfeeding (OR 0.28, 95% CI 0.21, 0.38) and an urban residence (OR 0.47, 95% CI 0.26, 0.86) had lower odds, while home birth had higher odds (OR 3.93, 95% CI 2.17, 7.10) of prelacteal feeding in Ethiopia.

Conclusions

In Ethiopia, one in four children were given prelacteal foods. Mothers who gave birth at home are more prone to give prelacteal foods. Whereas, antenatal care, timely initiation of breastfeeding, counseling on infant feeding and an urban residence decreases prelacteal feeding practices in Ethiopia. Therefore, the government and health institutions should focus to increase maternal health service utilization and promote infant and young child feeding practices according to the guideline.