Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F₁ ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and β_DP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.     

Ce−ZSM-5: A Highly Capable Catalyst for the Construction of Acridines in Water and Their Computational Study against DNA Gyrase Protein

Cerium doped ZSM-5 (Ce−ZSM-5) as an environmentally benign and reusable catalyst for the construction of acridines in aqueous medium. This method produced corresponding acridines with good yields and shorter reaction times. Also avoids the use of hazardous solvents and involves a simple work-up process. The solid catalyst was prepared by doping Ce ions with ZSM-5 (Zeolite Socony Mobil-5) and confirmed by XRD, BET SA-PSD and SEM. The synthesised acridine derivatives were confirmed by ¹H-NMR, ¹³C-NMR and FT-IR spectroscopic data. The docking studies of the synthesised compounds are performed by the PyRx auto dock tool against DNA gyrase protein. The products 5a and 6d are found to be the best fit ligands against DNA gyrase protein.     

Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae

The mitochondrial ATP synthase from yeast S. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. The ATP synthase has been modified on the N-terminus of the β-subunit to include a His(6) tag for Ni-chelate affinity purification. The enzyme is purified by a two-step procedure from submitochondrial particles and the resulting enzyme demonstrates lipid dependent oligomycin sensitive ATPase activity of 50 units/mg. The yeast ATP synthase shows a strong lipid selectivity, with cardiolipin (CL) being the most effective activating lipid and there are 30 moles CL bound per mole enzyme at saturation. Green Fluorescent Protein (GFP) has also been fused to the C-terminus of the ε-subunit to create a steric block for rotation of the central stalk. The ε-GFP fusion peptide is imported into the mitochondrion, assembled with the ATP synthase, and inhibits ATP synthetic and hydrolytic activity of the enzyme. F(1)F(o) ATP synthase with ε-GFP was purified to homogeneity and serves as an excellent enzyme for two- and three-dimensional crystallization studies.     

Oviposition Site-Selection by Bactrocera dorsalis Is Mediated through an Innate Recognition Template Tuned to γ-Octalactone

Innate recognition templates (IRTs) in insects are developed through many years of evolution. Here we investigated olfactory cues mediating oviposition behavior in the oriental fruit fly, Bactrocera dorsalis, and their role in triggering an IRT for oviposition site recognition. Behavioral assays with electrophysiologically active compounds from a preferred host, mango, revealed that one of the volatiles tested, γ-octalactone, had a powerful effect in eliciting oviposition by gravid B. dorsalis females. Electrophysiological responses were obtained and flies clearly differentiated between treated and untreated substrates over a wide range of concentrations of γ-octalactone. It triggered an innate response in flies, overriding inputs from other modalities required for oviposition site evaluation. A complex blend of mango volatiles not containing γ-octalactone elicited low levels of oviposition, whereas γ-octalactone alone elicited more oviposition response. Naïve flies with different rearing histories showed similar responses to γ-octalactone. Taken together, these results indicate that oviposition site selection in B. dorsalis is mediated through an IRT tuned to γ-octalactone. Our study provides empirical data on a cue underpinning innate behavior and may also find use in control operations against this invasive horticultural pest.     

Approaches to the simultaneous inactivation of metallo- and serine-β-lactamases

A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-β-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-β-lactamase. With respect to interactions with the classes A, C, and D serine β-lactamases, as judged by their correspondingly low K_m values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N–H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine β-lactamases, the OC–NR–OH functionality can function as a structural isostere of the OC–NR–H group, with the N–O–H group presumably replacing the amide N–H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k_cat values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine β-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N–H bond by N–OH may represent a useful strategy for the inhibition of other serine hydrolases.     

Short-term exercise reduces markers of hepatocyte apoptosis in nonalcoholic fatty liver disease

Increased hepatocyte apoptosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and contributes to the profibrogenic state responsible for the progression to nonalcoholic steatohepatitis (NASH). Strategies aimed at reducing apoptosis may result in better outcomes for individuals with NAFLD. We therefore examined the effect of a short-term exercise program on markers of apoptosis-plasma cytokeratin 18 (CK18) fragments, alanine aminotransferase (ALT), aspartate aminotransferase (AST), soluble Fas (sFas), and sFas ligand (sFasL)-in 13 obese individuals with NAFLD [body mass index 35.2 ± 1.2 kg/m(2), >5% intrahepatic lipid (IHL) assessed by (1)H-MR spectroscopy]. Exercise consisted of treadmill walking for 60 min/day on 7 consecutive days at ～85% of maximal heart rate. Additionally, subjects underwent an oral glucose tolerance test and a maximal oxygen consumption (Vo(2max)) test before and after the exercise intervention. The Matsuda index was used to assess insulin sensitivity. We observed significant decreases in CK18 fragments (558.4 ± 106.8 vs. 323.4 ± 72.5 U/l, P < 0.01) and ALT (30.2 ± 5.1 vs. 24.3 ± 4.8 U/l, P < 0.05), and an increase in whole body fat oxidation (49.3 ± 6.1 vs. 69.4 ± 7.1 mg/min, P < 0.05), while decreases in circulating sFasL approached statistical significance (66.5 ± 6.0 vs. 63.0 ± 5.7 pg/ml, P = 0.06), as did the relationship between percent change in circulating CK18 fragments and ALT (r = 0.55, P = 0.05). We also observed a significant correlation between changes in fat oxidation and circulating sFasL (rho = -0.65, P < 0.05). There was no change in IHL following the intervention (18.2 ± 2.5 vs. 17.5 ± 2.1%, NS). We conclude that short-term exercise reduces a circulatory marker of hepatocyte apoptosis in obese individuals with NAFLD and propose that changes in the proapoptotic environment may be mediated through improved insulin sensitivity and increased oxidative capacity.     

Phylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl–CoA-O- methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala)

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 α-helices, 6 and 7 β-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.     

Effect of Moringa oleifera leaves on hematological profile of fluorosis affected rats

Fluorosis is a metabolic disease that is endemic in nearly 25 countries with India being one of the most affected. It primarily affects the bone and the teeth. Moringa oleifera (MO) leaves are known to reduce the effect of fluorosis on various tissues. Therefore, it is of interest to document the effect of Moringa oleifera leaves on the hematological profile of fluorosis affected rats. Twenty four Sprague Dawley rats were housed two per cage in a room with 12 hours light and 12 hours dark cycle. The rats were allowed to adjust to the laboratory environment for about one to two weeks before the beginning of the study. This study reveals that MO leaves is effective in reducing the plasma fluoride content. It also helps in improving the Hb % and RBC count in fluorosis affected rats. Data shows that Moringa olifera leaves powder is effective in reducing the plasma fluoride content. It also helps in improving the Hemoglobin percentage & Red Blood Cell count in fluorosis affected rats.     

The Growing Season,but Not the Farming System,Is a Food Safety Risk Determinant for Leafy Greens in the Mid-Atlantic Region of the United States

Small- and medium-size farms in the mid-Atlantic region of the United States use varied agricultural practices to produce leafy greens during spring and fall, but the impact of preharvest practices on food safety risk remains unclear. To assess farm-level risk factors, bacterial indicators, Salmonella enterica, and Shiga toxin-producing Escherichia coli (STEC) from 32 organic and conventional farms were analyzed. A total of 577 leafy greens, irrigation water, compost, field soil, and pond sediment samples were collected. Salmonella was recovered from 2.2% of leafy greens (n = 369) and 7.7% of sediment (n = 13) samples. There was an association between Salmonella recovery and growing season (fall versus spring) (P = 0.006) but not farming system (organic or conventional) (P = 0.920) or region (P = 0.991). No STEC was isolated. In all, 10% of samples were positive for E. coli: 6% of leafy greens, 18% of irrigation water, 10% of soil, 38% of sediment, and 27% of compost samples. Farming system was not a significant factor for levels of E. coli or aerobic mesophiles on leafy greens but was a significant factor for total coliforms (TC) (P < 0.001), with higher counts from organic farm samples. Growing season was a factor for aerobic mesophiles on leafy greens (P = 0.004), with higher levels in fall than in spring. Water source was a factor for all indicator bacteria (P < 0.001), and end-of-line groundwater had marginally higher TC counts than source samples (P = 0.059). Overall, the data suggest that seasonal events, weather conditions, and proximity of compost piles might be important factors contributing to microbial contamination on farms growing leafy greens.