Distribution of ABCB1, CYP3A5, CYP2C19, and P2RY12 gene polymorphisms in a Mexican Mestizos population

The aim of the present study was to establish the gene frequency of six polymorphisms of the ABCB1, CYP3A5, CYP2C19, and P2RY12 genes in a population resident of Mexico City. The proteins encoded by these genes have been associated with the absorption, and biotransformation of clopidogrel. The ABCB1 T3435C, CYP3A5 V3* A6986G, P2RY12 G52T, P2RY12 C34T, CYP2C19 V2* and V3* (positions G681A and G636A, respectively), polymorphisms were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 269 healthy unrelated Mexican Mestizo individuals. The CYP2C19 V3* G636A polymorphism was not detected in the Mexican Mestizos population. However, the studied population presented significant differences (P < 0.05) in the distribution of the T3435C, A6986G, G681A, G52T and C34T polymorphisms when compared to reported frequencies of Amerindian of South America, Caucasian, Asian, and African populations. In summary, the distribution of the ABCB1, CYP3A5, CYP2C19, and P2RY12 gene polymorphisms distinguishes to the Mexican Mestizos population from other ethnic groups.     

Association of polymorphic markers of chemokine genes,their receptors,and <Emphasis Type="Italic">CD14</Emphasis> gene with coronary atherosclerosis

Atherosclerosis represents an inflammatory response to the disturbance of the endothelial layer in the arterial bloodstream. In the present study, an analysis of associations of polymorphic markers for the genes controlling synthesis of proteins involved in atherosclerosis pathogenesis in coronary atherosclerosis (CA) patients (217 subjects) and in a control group (250 subjects) was conducted. The following genes were examined: rs991804 (CCL2 gene), rs1126579 (CXCR2 gene), rs4074 (CXCL1 gene), rs4073 (CXCL8 gene), rs333 (CCR5 gene), rs2471859 (CXCR4 gene), rs1801157 (CXCL12 gene), and rs2569190 (CD14 gene). Using the Monte Carlo and Markov chain (APSampler) method, allele/genotype combinations associated with both low and high CA risk were revealed. The most important findings included the following: CXCR4*T/T + CCL2*C + CCR5*I/I (P_perm = 1 × 10^–6, OR = 0.44, 95% CI 0.3–0.63), CXCR2*C + CD14*C + CXCL12*G + CCL2*C + CCR5*D (P_perm = 4 × 10^–6, OR = 5.78, 95% CI 2.34–14.28), CD14*C + CCL2*C/C + CCR5*D (P_perm = 6.3 × 10^–6, OR = 5.81, 95% CI 2.17–15.56), CXCL8*A + CXCR2*C + CD14*T + CXCR4*C (P_perm = 0.01, OR = 3.21, 95% CI 1.63–6.31).     

Identification of Stringent Response-Related and Potential Serological Proteins Released from Bacillus anthracis Overexpressing the RelA/SpoT Homolog,Rsh Bant

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh _Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh _Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh _Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh _Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh _Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.     

Immune response of mice to the Thy-1.1 antigen: Effect of theIr-5 alleles studied in 129/J and B10.129 (6M) mice

The primary immune response to the Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes. B10.129(6M) mice carrying theH-2 complex of an intermediate responder (129) on a low-responder (B10) background, were low responders. Studies employing different F₁ hybrids and segregating generations of 129/J and 6M mice indicated that differences in responsiveness of those two strains depend on alleles at a single locus, loosely linked to theH-2 complex. These results lend further support to the previously advanced concept that the expression of theIr-Thy-1 allcles controlling the response to the Thy-1.1 antigen is influenced by the alleles at theIr-5 locus. In addition, studies employing F₁ hybrids produced through matings of 129/J, 6M, C3H.B10 and C57BL/6J mice to a panel of inbred strains suggested that in regard to the responsiveness to the Thy-1.1 antigen, 129/J and 6M mice are phenotypically, and presumably genotypically, similar to C3H.B10 and C57BL/6J mice, respectively.     

Susceptibility to db gene and streptozotocin-induced diabetes in C57BL mice: control by gender-associated,MHC-unlinked traits

H-2 haplotype differences distinguish the related C57BL/KsJ (BKs) and C57BL/6J (B6) inbred strains. BKs mice are more susceptible to diabetes induction by a recessive obesity gene, diabetes (db), or by multi-dose streptozotocin (MSZ) administration. The purpose of this study was to evaluate whether the H-2 differences were the important genetic background modifiers determining inbred strain susceptibility or resistance to these diabetogenic stresses. Diabetes susceptibility of BKs.B6-H-2 ^b congenic mice was compared with that of the parental BKs and B6 stocks. In addition, diabetes severity was studied in (B6 × BKs)F₁ and F₂ db/db mice and an H-2 segregation analysis was performed. BKs susceptibility genes expressed in a dominant fashion in the F₁ generation, and were transmitted to F₂ db/db males without apparent segregation. No association between H-2 ^b haplotype and B6-type diabetes resistance was found in response to either the db mutation or to MSZ. Insulitis, associated with development of hyperglycemia in BKs males, also occurred in the H-2 ^b congenic stock. However, an apparent interaction between H-2 ^b haplotype, the db mutation (on chromosome 4), and male gender (Y chromosome?) was indicated by a segregation ratio distortion in recovery of this genotype. A more moderate diabetes in some F₂ db/db females suggested that non-MHC-linked genes controlling sex steroid metabolism were the important determinants of diabetogenic sensitivities in the C57BL stocks. In support of the latter, strain differences were demonstrated in activity levels of steroid sulfatase, which is regulated by a sex-linked gene likely expressed on both the X and Y chromosome, and which may control tissue levels of active androgens and estrogens. We show that the diabetes-susceptible F₁ hybrids exhibit the higher activity associated with the BKs strain.     

Bacterial community analysis in chlorpyrifos enrichment cultures via DGGE and use of bacterial consortium for CP biodegradation

The organophosphate pesticide chlorpyrifos (CP) has been used extensively since the 1960s for insect control. However, its toxic effects on mammals and persistence in environment necessitate its removal from contaminated sites, biodegradation studies of CP-degrading microbes are therefore of immense importance. Samples from a Pakistani agricultural soil with an extensive history of CP application were used to prepare enrichment cultures using CP as sole carbon source for bacterial community analysis and isolation of CP metabolizing bacteria. Bacterial community analysis (denaturing gradient gel electrophoresis) revealed that the dominant genera enriched under these conditions were Pseudomonas, Acinetobacter and Stenotrophomonas, along with lower numbers of Sphingomonas, Agrobacterium and Burkholderia. Furthermore, it revealed that members of Bacteroidetes, Firmicutes, α- and γ-Proteobacteria and Actinobacteria were present at initial steps of enrichment whereas β-Proteobacteria appeared in later steps and only Proteobacteria were selected by enrichment culturing. However, when CP-degrading strains were isolated from this enrichment culture, the most active organisms were strains of Acinetobacter calcoaceticus, Pseudomonas mendocina and Pseudomonas aeruginosa. These strains degraded 6–7.4 mg L^?1 day^?1 of CP when cultivated in mineral medium, while the consortium of all four strains degraded 9.2 mg L^?1 day^?1 of CP (100 mg L^?1). Addition of glucose as an additional C source increased the degradation capacity by 8–14 %. After inoculation of contaminated soil with CP (200 mg kg^?1) disappearance rates were 3.83–4.30 mg kg^?1 day^?1 for individual strains and 4.76 mg kg^?1 day^?1 for the consortium. These results indicate that these organisms are involved in the degradation of CP in soil and represent valuable candidates for in situ bioremediation of contaminated soils and waters.     

Fumonisin B1: Isolation from corn culture,and purification by high performance liquid chromatography

A method is described to isolate fumonisin B₁ (FB₁) from corn cultured for 18 days at 25°C withFusarium moniliforme. Cultured corn was extracted with aqueous methanol and purified with XAD-2 column chromatography and high performance liquid chromatography (HPLC). About 450 mg of FB₁ were obtained from 800g cultured corn. Its identity was established by fast-atom bombardment (FAB) mass spectrometry, and infrared spectrum and nuclear magnetic spectrum. Its purity was estimated to be 95% by gas chromatography/mass spectrometry (GC/MS).References     

Esterase-16 (Es-16): Characterization,polymorphism, and linkage to chromosome 3 of a kidney esterase locus of the house mouse

A polymorphism for an isozyme of a presumed arylesterase, esterase-16 (EC 3.1.1.2), has been detected in kidney, heart, and spleen of the house mouse, Mus musculus, by means of isoelectric focusing and by disc electrophoresis. Three phenotypes can be distinguished: the ES-16A phenotype (IEP 5.9) was found in C57BL/10Sn and many other laboratory inbred strains; the ES-16B phenotype (IEP 6.1) was found in M. m. molossinus; and the ES-16C phenotype (IEP 5.9; very weak activity) was found in Peru-Coppock. Esterase-16 is strongly inhibited by 10^?3 m p-chloromercuribenzoate, but not by 2·10^?4 m bis-p-nitrophenyl phosphate or by 10^?3 m Diamox. It stains well with indoxyl acetate and other indigogenic substrates but only weakly with α-naphthyl acetate. Esterase-16 is completely insoluble in water. It is apparently governed by a structural gene locus, Es-16, with three alleles, Es-16 ^a , Es-16^b, and Es-16 ^c, respectively. Es-16 is closely linked to Car-1 and Car-2 on chromosome 3. Typing of 94 animals of the backcross (C57BL/10Sn × M. m. mol.) F₁ × M. m. mol. revealed a recombination frequency of 8.51±2.9%.     

Genetic variation in the activity of the histidine catabolic enzymes between inbred strains of mice: A structural locus for a cytosol histidine aminotransferase isozyme (Hat-1)

Variation in activity of the main histidine catabolic enzymes (histidase, urocanase, and aminotransferase) has been surveyed using inbred strains of mice (C57BL, DBA, Peru, SM, and SWR). Some variation was found in the activity of all enzymes, but only in the case of cytosolic histidine aminotransferase was it greater than twofold (SM 3.3-fold greater than C57BL). The divergent strains for the activity of this enzyme were crossed and the F ₁ 's were backcrossed; the segregation analysis indicated a single locus with additively acting alleles (designated Hat-1: a allele SM, b allele C57BL). Cytosolic histidine aminotransferase differed in heat stability between SM and C57BL, indicating that Hat-1 is a structural locus. The conflict in the biochemical literature (Morris et al., 1973; Noguchi et al., 1976a, b) over the number and subcellular distribution of the histidine aminotransferase isozymes is partly resolved by the acquisition of a variant at the Hat-1 locus. Hat-1 affects the cytosolic form but not the mitochondrial form of the enzyme. Purification and analysis of the isozymes of histidine aminotransferase from livers of C57BL and SM mice will further clarify the situation.     

Affinity labeling of the active site of pig liver NADH-cytochrome b5 reductase by 5′-p-fluorosulfonylbenzoyladenosine

Lysosome-solubilized pig liver NADH-cytochrome b₅ reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/K _obs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.K _d or the initial reversible enzyme-analogue complex is estimated at 185 µM withK ₂=0.22 min^?1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[¹⁴C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b₅ reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme.     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Four mononuclear platinum(II) complexes: synthesis,DNA/BSA binding,DNA cleavage and cytotoxicity

Four new platinum(II) complexes: Pt^II L1·H₂O (C1, H₂ L1 = C₂₀H₁₆N₂O₂), Pt^II L2Cl₂ (C2, L2 = C₂₂H₁₆N₂O₂), Pt^II L3Cl₂·H₂O (C3, L3 = C₂₀H₁₆N₂), Pt^II L4Cl₂·0.4H₂O (C4, L4 = C₁₈H₁₄N₄) have been synthesized and characterized by using various physico-chemical techniques. The binding interaction of the four platinum(II) complexes C1–C4 with calf thymus (CT)-DNA has been investigated by UV–Vis and fluorescence emission spectrometry. The apparent binding constant (K _app) values follow the order: C3 > C1 > C2 > C4. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the four platinum(II) complexes C1–C4 showed that the quenching mechanism might be a static quenching procedure. For C1–C4, the number of binding sites was about one for BSA and the binding constants follow the order: C3 (7.08 × 10⁵M^?1) > C1 (2.82 × 10⁵M^?1) > C2 (0.85 × 10⁵M^?1) > C4 (0.15 × 10⁵M^?1). With the single condition change such as absence of an external agent, the DNA cleavage abilities of C3 exhibit remarkable changes. In addition, the cytotoxicity of C3 in vitro on tumor cells lines (MCF-7, HepG2 and HT29) were examined by MTT and showed better antitumor effects on the tested cells.     

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: Codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1–6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F₁ hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1^b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1^a) mice were low responders. The V_H recombinant strains BAB.14 and CB-8KN that possess the Ig-1^b allotype of C57BL, but have some of theV _H genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.     

Cloning,expression and characterization of a novel aldehyde dehydrogenase gene from Flammeovirga pacifica

A novel putative aldehyde dehydrogenase (ALDH) gene aldh1413 from Flammeovirga pacifica isolated from deep sea sediment was cloned, expressed, and characterized. The molecular weight of the ALDH1413 (479 amino acids) was estimated by SDS-PAGE to be 53 kDa. The optimum temperature and pH for ALDH1413 were 35°C and 9.0, respectively. In the presence of either NAD⁺ or NADP⁺, the enzyme could oxidize a number of aliphatic aldehydes, particularly C3-and C5-aliphatic aldehydes and aromatic aldehydes such as benzaldehyde, which indicates that the enzyme belongs to broad-specific (ALDH) superfamily. Steady-state kinetic study revealed that ALDH1413 had a K _M value of 0.545 mM and a k _cat value of 7.48 s^?1 when propionaldehyde was used as the substrate. The Na⁺ could enhance ALDH1413 activity, which indicated it might be adapt to its habitat, marine environment.