Escherichia coli membrane-derived oxygen-reducing enzyme system (Oxyrase) protects bubaline spermatozoa during cryopreservation.

The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane-derived oxygen scavenger (Oxyrase) on post-thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris-egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post-thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo-3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine-phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F-pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose-dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase-fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose-dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post-thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

Genetic variants in BCMO1 and CD36 are associated with plasma lutein concentrations and macular pigment optical density in humans.

UNLABELLED: Lutein is recovered at high concentration in the human macula lutea. Recent studies suggest that this micronutrient might be implicated in prevention of age-related macular degeneration. OBJECTIVE: to identify genes which affect blood and retina lutein concentrations among candidate genes (intestinal sterol transporters and carotenoid oxygenases). DESIGN: a comparative plus an observational study. PARTICIPANTS: twenty-nine healthy subjects for the comparative study and 622 subjects for the observational study. INTERVENTION AND METHODS: all the participants were genotyped for single nucleotide polymorphisms (SNPs) in the candidate genes. Fasting plasma lutein concentrations were measured in all the participants and after 6 months' supplementation, with either a lutein-rich supplement or a placebo, in the 29 subjects who participated in the comparative study. Macular pigment optical density (MPOD), which is a measure of macula concentration of lutein, was measured before and after the dietary intervention in the 29 subjects. Associations between SNPs and plasma lutein and MPOD were assessed by partial least square (PLS) regression followed by univariate analysis. Observed associations between SNPs and plasma lutein were verified by haplotype-based association analysis in the cohort of 622 subjects. MAIN OUTCOME MEASURES: plasma lutein levels and MPOD. RESULTS: six SNPs in four genes (ABCG8, BCMO1, CD36, and NPC1L1) explained 25% and 38% of the plasma and MPOD variance, respectively. Subjects with TT at the BCMO1 rs7501331 locus had lower (P < 0.05) plasma lutein than CT subjects. Subjects with CC at the CD36 rs13230419 locus had lower (P < 0.05) plasma lutein than subjects who carried a T allele. The association between CD36 and plasma lutein was confirmed in the cohort of 622 subjects. Subjects with TT at the BCMO1 rs7501331 locus had a higher (P < 0.05) MPOD, and subjects with GG at rs1761667 CD36 locus had a higher (P < 0.05) MPOD than those with an A allele. CONCLUSIONS: these results suggest that BCMO1 and CD36 are implicated in plasma and retina concentrations of lutein and that genetic variants in these genes can modulate blood and retina concentrations of lutein.

Complex mechanism underlying transcriptional control of the haplotyped flavin-containing monooxygenase 3 (FMO3) gene in Japanese: different regulation between mutations in 5'-upstream distal region and common element in proximal region.

We reported the human flavin-containing monooxygenase 3 (FMO3) haplotypes (Pharmacogenet. Genomics: 17, 827, 2007). The objective was to gain the insight into transcriptional regulation in a Japanese population. The wild-type FMO3 reporter plasmids carrying 5'-flanking sequence from the transcriptional initiation site of the FMO3 haplotype 1 (prepared from three individuals) showed higher luciferase activities in HepG2 cells than those from the FMO3 haplotypes 2 and 3, with the wild-type coding region. Several deletion mutants of the FMO3 haplotype 1 (extending from -5,167 to -1,764, numbered relative to the A of the ATG translational initiation codon) revealed that the region of -2,064 to -1,804 contained an important cis-acting element(s) for activation of the FMO3 gene expression. Putative hepatocyte nuclear factor-4 (HNF-4) binding site and CCAAT box, but not Yin Yang 1 element, could be responsible cis-acting elements of the FMO3 gene, by site-directed mutagenesis analysis. The unknown suppressive cis-element(s) at the 5'-upstream region from -2,064 might show genetic polymorphism, because the FMO3 haplotypes 2 and 3 had three and ten mutations, respectively. These results suggest that the putative HNF-4 binding site and CCAAT box could be responsible cis-acting elements of the FMO3 gene in Japanese.

Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia.

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.

Genetic variability at the human FMO1 locus: significance of a basal promoter yin yang 1 element polymorphism (FMO1*6).

The flavin-containing monooxygenases (FMOs) are important for the disposition of a variety of toxicants, therapeutics, and dietary components. Although FMO1 is the dominant isoform in fetal liver and adult kidney and intestine and despite up to a 10-fold intersubject variation in expression, a paucity of information is available on FMO1 genetic variability. To address this issue, 24 samples from the Coriell DNA Polymorphism Discovery Resource Panel were sequenced revealing 10 common single nucleotide polymorphisms (SNPs): four located upstream of the structural gene; three within exonic sequences; one within the intron 1 splice donor site; and two with the 3'-untranslated region. Six of these variants are novel. Compared with other FMO loci within the chromosome 1q23-25 cluster, FMO1 seems more highly conserved. Of the identified FMO1 SNPs, only a C>A transversion 9536 base pairs upstream of the exon 2 ATG start codon (g.-9536C>A) would likely affect function, because it lies within the conserved core binding sequence for the yin yang 1 (YY1) transcription factor. Electrophoretic mobility shift assays demonstrated that the g.-9536C>A transversion eliminated YY1 binding. Furthermore, data from transient expression assays in HepG2 cells suggested this SNP could account for a 2- to 3-fold loss of FMO1 promoter activity. Genotype analysis revealed a g.-9,536A allele (FMO1*6) frequency of 13 and 11% in African- and northern European-Americans, respectively, but a significantly higher frequency of 30% in Hispanic-Americans. Thus, the FMO1*6 variant may account for some of the observed interindividual variation in FMO1 expression.

Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria.

Trimethylaminuria is a rare metabolic disorder that is associated with abnormal amounts of the dietary-derived trimethylamine. Excess unmetabolized trimethylamine in the urine, sweat and other body secretions confers a strong, foul body odor that can affect the individual's ability to work or engage in social activities. This review summarizes the biochemical aspects of the condition and the classification of the disorder into: 1) primary genetic form, 2) acquired form, 3) childhood forms, 4) transient form associated with menstruation, 5) precursor overload and 6) disease states. The genetic variability of the flavin-containing monooxygenase (form 3) that is responsible for detoxication and deodoration of trimethylamine is discussed and put in context with other variant forms of the flavin-containing monooxygenase (forms 1-5). The temporal-selective expression of flavin-containing monooxygenase forms 1 and 3 is discussed in terms of an explanation for childhood trimethylaminuria. Information as to whether variants of the flavin-containing monooxygenase form 3 contributes to hypertension and/or other diseases are presented. Discussion is provided outlining recent bioanalytical approaches to quantify urinary trimethylamine and trimethylamine N-oxide and plasma choline as well as data on self-reporting individuals tested for trimethylaminuria. Finally, trimethylaminuria treatment strategies and nutritional support are described including dietary sources of trimethylamine, vitamin supplementation and drug treatment and issues related to trimethylaminuria in pregnancy and lactation are discussed. The remarkable progress in the biochemical, genetic, clinical basis for understanding the trimethylaminuria condition is summarized and points to needs in the treatment of individuals suffering from trimethylaminuria.

Contribution of flavin-containing monooxygenase and cytochrome P450 to imipramine N-oxidation in rat hepatic microsomes.

Enzymatic formation of desipramine (DMI) and imipramine N-oxide (IMINO) was kinetically characterized in rat liver microsomes at pH 8.5 and 7.5. The formation of IMINO was quickly suppressed by the preincubation of microsomes at 37 degrees C at pH 8.5, but the suppression was comparatively gentle at pH 7.5. In kinetic studies, the formation of DMI was monophasic at the two pH points, and a substrate inhibition was observed at pH 8.5, but not at pH 7.5. In contrast, the formation of IMINO was biphasic at both pH points, Le., the summation of a low-Km phase and a high-Km phase. Methimazole (MZ), an inhibitor of flavin-containing monooxygenase (FMO), markedly suppressed the low-Km phase of IMINO formation at both pH points. MZ also suppressed DMI formation at pH 8.5, but it elevated DMI formation at pH 7.5. SKF 525-A, an inhibitor of cytochrome P450 (CYP), markedly suppressed DMI formation at both pH points. The inhibitor suppressed IMINO formation in the high-Km phase of the biphasic kinetics at both pH points, whereas it stimulated the activity of the low-Km phase at pH 7.5. These results suggest that CYP enzyme(s) are mainly responsible for DMI formation at pH 8.5 and 7.5, and FMO enzyme(s) also are involved in IMI N-demethylation at a higher pH range in rat liver microsomes, at least in part. In the formation of IMINO, FMO is a major enzyme at both pH points, and CYP may also contribute to the N-oxide formation to some extent at pH 8.5.

PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes.

A method was developed to isolate central segments of catechol 2, 3-dioxygenase (C23O) genes from environmental samples and to insert these C23O gene segments into nahH (the structural gene for C23O encoded by catabolic plasmid NAH7) by replacing the corresponding nahH sequence with the isolated segments. To PCR-amplify the central C23O gene segments, a pair of degenerate primers was designed from amino acid sequences conserved among C23Os. Using these primers, central regions of the C23O genes were amplified from DNA isolated from a mixed culture of phenol-degrading or crude oil-degrading bacteria. Both the 5' and 3' regions of nahH were also PCR-amplified by using appropriate primers. These three PCR products, the 5'-nahH and 3'-nahH segments and the central C23O gene segments, were mixed and PCR-amplified again. Since the primers for the amplification of the central C23O gene segments were designed so that the 20 nucleotides at both ends of the segments are identical to the 3' end of the 5'-nahH segment and the 5' end of the 3'-nahH segment, respectively, the central C23O gene segments could anneal to both the 5'- and 3'-nahH segments. After the second PCR, hybrid C23O genes in the form of (5'-nahH segment-central C23O gene segment-3'-nahH segment) were amplified to full length. The resulting products were cloned into a vector and used to transform Escherichia coli. This method enabled divergent C23O sequences to be readily isolated, and more than 90% of the hybrid plasmids expressed C23O activity. Thus, the present method is useful to create, without isolating bacteria, a library of functional hybrid genes.