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Chronic arsenic exposure via drinking water has become a worldwide public health concern. In humans, inorganic arsenic (iAs) is metabolized to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) mainly mediated by arsenic (þ3 oxidation state) methyltransferase (As3MT). We reported recently that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) was involved in arsenic metabolism, and examined its interactive effect with As3MT on arsenic metabolism in vitro. To further evaluate the interactive effect of N6AMT1 and As3MT on arsenic biomethylation in humans, we conducted a human population-based study including 289 subjects living in rural villages in Inner Mongolia, China, and assessed their urinary arsenic metabolites profiles in relation to genetic polymorphisms and haplotypes of N6AMT1 and As3MT. Five N6AMT1 single nucleotide polymorphisms (SNPs; rs1003671, rs7282257, rs2065266, rs2738966, rs2248501) and the N6AMT1 haplotype 2_GGCCAT were significantly associated with the percentage of iAs (% iAs) in urine (e.g., for rs7282257, mean was 9.62% for TT, 6.73% for AA). Rs1003671 was also in a significant relationship with urinary MMA and DMA (the mean of %MMA was 24.95% for GA, 31.69% for GG; the mean of % DMA was 69.21% for GA, 59.82% for GG). The combined effect of N6AMT1 haplotype 2_GGCCAT and As3MT haplotype 2_GCAC showed consistence with the additive significance of each haplotype on % iAs: the mean was 5.47% and 9.36% for carriers with both and null haplotypes, respectively. Overall, we showed that N6AMT1 genetic polymorphisms were associated with arsenic biomethylation in the Chinese population, and its interaction with As3MT was observed in specific haplotype combinations. [ABSTRACT FROM AUTHOR]

We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway. [ABSTRACT FROM AUTHOR]

Drosomycin (Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes, Dro1, Dro2, Dro3, Dro4, Dro5, and Dro6, forming a multigene family on the 3L chromosome arm in Drosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the seven drosomycin genes, Drs, Dro2, Dro3, Dro4, and Dro5 showed constitutive expressions. Three out of five, Dro2, Dro3, and Dro5, were able to be upregulated by simple injury. Interestingly, Drs is an only gene strongly upregulated when Drosophila was infected with microbes. In contrast to these five genes, Dro1 and Dro6 were not transcribed at all in either noninfected or infected flies. Furthermore, by 5′ rapid amplification of cDNA ends, two transcription start sites were identified in Drs and Dro2, and one in Dro3, Dro4, and Dro5. In addition, NF-κB binding sites were found in promoter regions of Drs, Dro2, Dro3, and Dro5, indicating the importance of NF-κB binding sites for the inducibility of drosomycin genes. Based on the analyses of flanking sequences of each gene in D. melanogaster and phylogenetic relationship of drosomycins in D. melanogaster species-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates of drosomycin genes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes. [ABSTRACT FROM AUTHOR]

The most economically significant Chinese cotton cultivar ( Gossypium hirsutum L. cv. Zhongmian 35) was transformed via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast-transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium containing glyphosate. Thirty-four independent transgenic lines were obtained after selection, giving a maximal 1.9% transformation frequency. The integration and expression of the aroA-M1 gene in T0 plants and T1 progeny were confirmed using DNA hybridization, Western blot and PCR techniques. An increased resistance of T0 and T1 transgenic plants towards glyphosate was also observed. [ABSTRACT FROM AUTHOR]

Pathogenesis-related proteins (PR), including β-1,3-glucanases may provide the first line of defense against fungal pathogens. Many PR proteins are activated by salicylic acid (SA), which acts as an endogenous signal. We have previously isolated seven members of the β-1,3-glucanase gene family in barley ( Hordeum vulgare). In this paper, we characterized the β-1,3-glucanase isoenzyme GIII for SA-responsive elements in the GIII gene promoter. A series of deletion mutations of the promoter were fused to the reporter gene beta-glucuronidase ( gus). The GUS activity was analyzed in rice calli ( Oryza sativa L.) in response to SA. A deletion fragment between −362 and +106 bp showed the highest level of GUS activity in these assays. This promoter fused with gus was further introduced into rice plants for stable transformation. Histochemical staining and fluorometric quantitation of GUS activity in leaves of transgenic plants revealed prominent GUS expression after SA induction. RNA analysis by Northern blotting confirmed the importance of this region, indicating that cis-acting elements required for SA-inducible expression exist within 362 bp upstream from the transcriptional start site. [ABSTRACT FROM AUTHOR]

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology. Selected mutant enzymes exhibited high Ki[glyphosate] and low Km[PEP] values compared to the parental enzymes from Escherichia coli (EcaroA) and Salmonella typhimurium (StaroA). One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco (Nicotiana tabacum cv. Xanthi). Transgenic plants with increased resistance to glyphosate were generated. [ABSTRACT FROM AUTHOR]

Reports on a study which reveals that a T42M substitution in bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) generates enzymes with increased resistance to glyphosate. Testimony for the powerful approach for protein engineering by the combination of directed evolution and rational design.

N-acetylchitooligosaccharides of degree of polymerization 6 to 8 elicit the synthesis of (1→3)-β-glucan endohydrolase (EC 3.2.1.39) activity in suspension-cultured cells derived from immature barley (Hordeum vulgare) embryos. Corresponding de-acetylated chitooligosaccharides have no effect. Concentrations of N-acetylchitoheptaose in the 200 nM range are sufficient to elicit the response. A 2- to 3-fold increase in (1→3)-β-glucanase activity is detected 24 h after the addition of the oligosaccharide. Non-denaturing gel electrophoresis, coupled with the in situ detection of (1→3)-β- glucanase activity in the gels, has enabled the separation of specific isoforms of the enzyme. The increase in (1→3)-β-glucanase activity following N-acetylchitoheptaose induction is attributable predominantly to enhanced levels of isoenzyme GII, although the barley (1→3)-β-glucanase gene family encodes at least seven different isoforms of the enzyme. Northern hybridization analyses with gene-specific probes continued the presence of mRNA encoding isoenzyme GII as the major mRNA in cells treated with the oligosaccharide elicitor. The results therefore demonstrate a specific induction of the (1→3)-β-glucanase isoenzyme Gil gene following stimulation of barley cells with oligosaccharides of fungal cell wall origin, and further suggest that a plant's response to microbial attack involves transcription of quite specific members of the gene families that encode pathogenesis-related proteins. [ABSTRACT FROM AUTHOR]