Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Promoters and processing sites within the transforming region of bovine papillomavirus type 1.

The mRNAs present in bovine papillomavirus type 1 (BPV-1)-transformed C127 cells were studied by primer extension. The results show that two internal promoters are present in the E region of BPV-1 in addition to the previously identified promoter at coordinate 1 (H. Ahola, A. Stenlund, J. Moreno-López, and U. Pettersson, Nucleic Acids Res. 11:2639-2650, 1983). One, located at coordinate 31, generated a set of mRNAs with heterogeneous 5' ends, which may encode the major transforming protein of BPV-1, the E5 protein. The second promoter, which is located at coordinate 39, generates colinear mRNAs which encode either the E4 protein or a truncated form of the E2 protein. Unlike the cottontail rabbit papillomavirus (O. Danos, E. Georges, G. Orth, and M. Yaniv, J. Virol. 53:735-741, 1985), BPV-1 appears to lack a separate promoter for expression of the E7 protein. The major splice sites in the transforming region (E region) of the BPV-1 genome were also identified by nucleotide sequence analysis.     

Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci

The aim of the work was the evaluation of different PCR-based methods to found an appropriate identification and typing strategy for environmental enterococci. Environmental enterococci were isolated mainly from surface- and waste-waters. Species identification was provided by combination of phenotypic (Micronaut System, Merlin) and molecular detection methods (fluorescent ITS-PCR, ddl-PCR, REP-PCR, AFLP). Very similar results were observed among molecular methods, however several discrepancies were recognized during comparison of molecular and biochemical identification. Seven enterococcal species (E. faecium, E. hirae, E. casseliflavus, E. mundtii, E. faecalis, E. durans and E. gallinarum) were identified within 166 environmental isolates. The results obtained in this work attest the importance of PCR-based methods for identification and typing of environmental enterococci. The fluorescent ITS-PCR (fITS-PCR) showed the best results in order to identify the enterococci strains, the method used the automated capillary electrophoresis to separate the PCR products in a very rapid and precise way. The AFLP method was suitable to identify and characterize the isolates, while the REP-PCR can be used for species identification.     

Synthesis and biological evaluation of some new A,B-ring modified steroidal D-lactones

Starting from the D-homo lactones of androst-4-en-3-one 3 and 4, prepared from 1 and 2, the new 17a homolactones 5-12, 14 and 15, were synthesized. The 4-hydroxy compounds 9 and 10 were obtained through the reaction of 4alpha,5alpha- (5 and 7) and 4beta,5beta- (6 and 8) epoxides with formic acid. The epoxides 5 and 6 were prepared from compound 3, and epoxides 7 and 8 from compound 4 by oxidation with H(2)O(2) under basic conditions. Compound 1 served as a starting substance for obtaining lactones 11-13. Oxidation of compound 1 with m-chloroperbenzoic acid yielded 11 and 12, but compound 13 gave 14. Compound 15 was obtained from 13 by oxidation with H(2)O(2) under basic conditions. The structures of epoxides 6 and 14 were confirmed by X-ray structural analysis. Cytotoxic activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Compounds 6 and 14 showed strong activity against PC3, the IC(50) being 10.6 and 2.2 microM, respectively, whereas compounds 3 and 8 showed strong activity against MDA-MB-231 (IC(50) is 9.3 and 3.6 microM, respectively). Aromatase inhibition assay showed that the tested compounds 9, 10, and 14 possess lower activity compared to formestane.     

A Combination of CD28 (rs1980422) and IRF5 (rs10488631) Polymorphisms Is Associated with Seropositivity in Rheumatoid Arthritis: A Case Control Study

IntroductionThe aim of the study was to analyse genetic architecture of RA by utilizing multiparametric statistical methods such as linear discriminant analysis (LDA) and redundancy analysis (RDA).MethodsA total of 1393 volunteers, 499 patients with RA and 894 healthy controls were included in the study. The presence of shared epitope (SE) in HLA-DRB1 and 11 SNPs (PTPN22 C/T (rs2476601), STAT4 G/T (rs7574865), CTLA4 A/G (rs3087243), TRAF1/C5 A/G (rs3761847), IRF5 T/C (rs10488631), TNFAIP3 C/T (rs5029937), AFF3 A/T (rs11676922), PADI4 C/T (rs2240340), CD28 T/C (rs1980422), CSK G/A (rs34933034) and FCGR3A A/C (rs396991), rheumatoid factor (RF), anti–citrullinated protein antibodies (ACPA) and clinical status was analysed using the LDA and RDA.ResultsHLA-DRB1, PTPN22, STAT4, IRF5 and PADI4 significantly discriminated between RA patients and healthy controls in LDA. The correlation between RA diagnosis and the explanatory variables in the model was 0.328 (Trace = 0.107; F = 13.715; P = 0.0002). The risk variants of IRF5 and CD28 genes were found to be common determinants for seropositivity in RDA, while positivity of RF alone was associated with the CTLA4 risk variant in heterozygous form. The correlation between serologic status and genetic determinants on the 1st ordinal axis was 0.468, and 0.145 on the 2nd one (Trace = 0.179; F = 6.135; P = 0.001). The risk alleles in AFF3 gene together with the presence of ACPA were associated with higher clinical severity of RA.ConclusionsThe association among multiple risk variants related to T cell receptor signalling with seropositivity may play an important role in distinct clinical phenotypes of RA. Our study demonstrates that multiparametric analyses represent a powerful tool for investigation of mutual relationships of potential risk factors in complex diseases such as RA.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. KI, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k3, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. KI values of 1.3 x 10(-4) M(-1), 5.6 x 10(-6) M(-1) and 7.2 x 10(-6)M(-1) were obtained for malathion, malaoxon and isomalathion, respectively. The IC50 values for free/immobilized AChE, (3.7 +/- 0.2) x 10(-4) M/(1.6 +/-0.1) x 10(-4), (2.4 +/- 0.3) x 10(-6)/(3.4 +/- 0.1) x 10(-6)M and (3.2 +/- 0.3) x 10(-6) M/(2.7 +/- 0.2) x 10(-6) M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 x 10(-7) M/2 x 10(-7) M, 2 x 10(-7) M/3 x 10(-7)M and 2 x 10(-7) M/4.5 x 109-7) M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Variance Heterogeneity in Saccharomyces cerevisiae Expression Data: Trans-Regulation and Epistasis

Here, we describe the results from the first variance heterogeneity Genome Wide Association Study (VGWAS) on yeast expression data. Using this forward genetics approach, we show that the genetic regulation of gene-expression in the budding yeast, Saccharomyces cerevisiae, includes mechanisms that can lead to variance heterogeneity in the expression between genotypes. Additionally, we performed a mean effect association study (GWAS). Comparing the mean and variance heterogeneity analyses, we find that the mean expression level is under genetic regulation from a larger absolute number of loci but that a higher proportion of the variance controlling loci were trans-regulated. Both mean and variance regulating loci cluster in regulatory hotspots that affect a large number of phenotypes; a single variance-controlling locus, mapping close to DIA2, was found to be involved in more than 10% of the significant associations. It has been suggested in the literature that variance-heterogeneity between the genotypes might be due to genetic interactions. We therefore screened the multi-locus genotype-phenotype maps for several traits where multiple associations were found, for indications of epistasis. Several examples of two and three locus genetic interactions were found to involve variance-controlling loci, with reports from the literature corroborating the functional connections between the loci. By using a new analytical approach to re-analyze a powerful existing dataset, we are thus able to both provide novel insights to the genetic mechanisms involved in the regulation of gene-expression in budding yeast and experimentally validate epistasis as an important mechanism underlying genetic variance-heterogeneity between genotypes.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.