Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.     

Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells

The presence ofa allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulateda3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypie determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-la antibodies. SDS-PAGE analyses of¹²⁵I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.     

Interaction of myotoxin a with artificial membranes: Raman spectroscopic investigation

Myotoxin a from the venom of Crotalus viridis viridis (prairie rattlesnake) is a small protein which is responsible for myonecrosis. It is a basic protein with 42 amino acid residues of known sequence. Three disulfide bonds give it a highly compact structure. Microscopic examination of the toxin's effects reveals that the most pronounced and earliest visible damage occurs intracellularly, in the sarcoplasmic reticulum membrane system of skeletal muscle. A better understanding of its mechanism of action is therefore of particular interest. The interaction of myotoxin a with artificial membranes (multibilamellar phospholipid dispersions) was investigated by using dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). Two regions of the Raman spectrum were examined for information: the C-H stretching region between 2800 and 3000 cm-1 and the C-C stretching region between 1000 and 1300 cm-1. The effects of myotoxin a on the thermotropic phase behavior of the artificial membranes were determined. This was done by monitoring three structurally sensitive Raman intensity ratios, I2932/2880, I2880/2850, and I1088/1126. It was found that myotoxin alpha destabilized the ordered structure of the gel phase of phospholipid bilayers. This effect was seen with both DMPC and DMPS. The pretransition of DMPC was perturbed by myotoxin a, while the main gel to liquid-crystal phase transition temperature was decreased. The effect of myotoxin a on the phase behavior of DMPS was found to be pH dependent with the least effect observed at low pH values. These results suggest the involvement of negatively charged phosphate groups of phospholipids in the interaction of myotoxin a with artificial membranes.     

福建省直播稻根结线虫病的病原种类鉴定

[目的] 福建省福清市江镜镇与福安市潭溪镇水稻区直播稻苗期分别发生严重的根结线虫病,本研究对其病原物进行形态和分子鉴定,明确病原物种类,以期为福建省直播稻根结线虫病害防控提供理论依据。[方法] 通过根结线虫各虫态形态学特征进行观测;同时对其rDNA-ITS区进行测序,通过贝叶斯法与最大似然法构建了系统发育树来确定种类;利用拟禾本科根结线虫特异性引物Mg-F/Mg-R检测种群。[结果] 根结线虫的雌虫、雄虫和2龄幼虫的形态学特征与拟禾本科根结线虫原始描述种一致;rDNA-ITS区序列长度为576 bp,与GenBank拟禾本科根结线虫种群相似度均达99%以上;系统发育树明确了该根结线虫与拟禾本科根结线虫处于同一分支;特异性引物Mg-F/Mg-R检测进一步明确病原为拟禾本科根结线虫单一种群。[结论] 本研究通过形态与分子特征,明确了在福建直播稻上发现的根结线虫为拟禾本科根结线虫。拟禾本科根结线虫在福建省最早于2011年在政和县小范围水稻田发现,此后未在其他水稻种植区发现。本次在福建直播稻上首次发现大面积的根结线虫为害。随着直播稻的种植面积扩大,拟禾本根结线虫引起的水稻病害可能会成为制约其发展的新问题,应引起足够重视。     

Genetic diversity of Trypanosoma cruzi parasites infecting dogs in southern Louisiana sheds light on parasite transmission cycles and serological diagnostic performance

BackgroundChagas disease is a neglected zoonosis of growing concern in the southern US, caused by the parasite Trypanosoma cruzi. We genotyped parasites in a large cohort of PCR positive dogs to shed light on parasite transmission cycles and assess potential relationships between parasite diversity and serological test performance.Methodology/principal findingsWe used a metabarcoding approach based on deep sequencing of T. cruzi mini-exon marker to assess parasite diversity. Phylogenetic analysis of 178 sequences from 40 dogs confirmed the presence of T. cruzi discrete typing unit (DTU) TcI and TcIV, as well as TcII, TcV and TcVI for the first time in US dogs. Infections with multiple DTUs occurred in 38% of the dogs. These data indicate a greater genetic diversity of T. cruzi than previously detected in the US. Comparison of T. cruzi sequence diversity indicated that highly similar T. cruzi strains from these DTUs circulate in hosts and vectors in Louisiana, indicating that they are involved in a shared T. cruzi parasite transmission cycle. However, TcIV and TcV were sampled more frequently in vectors, while TcII and TcVI were sampled more frequently in dogs.Conclusions/significanceThese observations point to ecological host-fitting being a dominant mechanism involved in the diversification of T. cruzi-host associations. Dogs with negative, discordant or confirmed positive T. cruzi serology harbored TcI parasites with different mini-exon sequences, which strongly supports the hypothesis that parasite genetic diversity is a key factor affecting serological test performance. Thus, the identification of conserved parasite antigens should be a high priority for the improvement of current serological tests.     

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.     

β3 Integrin Promotes TGF-β1/H2O2/HOCl-Mediated Induction of Metastatic Phenotype of Hepatocellular Carcinoma Cells by Enhancing TGF-β1 Signaling

In addition to being an important mediator of migration and invasion of tumor cells, β3 integrin can also enhance TGF-β1 signaling. However, it is not known whether β3 might influence the induction of metastatic phenotype of tumor cells, especially non-metastatic tumor cells which express low level of β3. Here we report that H₂O₂ and HOCl, the reactive oxygen species produced by neutrophils, could cooperate with TGF-β1 to induce metastatic phenotype of non-metastatic hepatocellular carcinoma (HCC) cells. TGF-β1/H₂O₂/HOCl, but not TGF-β1 or H₂O₂/HOCl, induced β3 expression by triggering the enhanced activation of p38 MAPK. Intriguingly, β3 in turn promoted TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of HCC cells by enhancing TGF-β1 signaling. β3 promoted TGF-β1/H₂O₂/HOCl-induced expression of itself via positive feed-back effect on p38 MAPK activation, and also promoted TGF-β1/H₂O₂/HOCl-induced expression of α3 and SNAI2 by enhancing the activation of ERK pathway, thus resulting in higher invasive capacity of HCC cells. By enhancing MAPK activation, β3 enabled TGF-β1 to augment the promoting effect of H₂O₂/HOCl on anoikis-resistance of HCC cells. TGF-β1/H₂O₂/HOCl-induced metastatic phenotype was sufficient for HCC cells to extravasate from circulation and form metastatic foci in an experimental metastasis model in nude mice. Inhibiting the function of β3 could suppress or abrogate the promoting effects of TGF-β1/H₂O₂/HOCl on invasive capacity, anoikis-resistance, and extravasation of HCC cells. These results suggest that β3 could function as a modulator to promote TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of non-metastatic tumor cells, and that targeting β3 might be a potential approach in preventing the induction of metastatic phenotype of non-metastatic tumor cells.     

Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition

Homer EVH1 (Ena/VASP Homology 1) domains interact with proline-rich motifs in the cytoplasmic regions of group 1 metabotropic glutamate receptors (mGluRs), inositol-1,4,5-trisphosphate receptors (IP3Rs), and Shank proteins. We have determined the crystal structure of the Homer EVH1 domain complexed with a peptide from mGluR (TPPSPF). In contrast to other EVH1 domains, the bound mGluR ligand assumes an unusual conformation in which the side chains of the Ser-Pro tandem are oriented away from the Homer surface, and the Phe forms a unique contact. This unusual binding mode rationalizes conserved features of both Homer and Homer ligands that are not shared by other EVH1 domains. Site-directed mutagenesis confirms the importance of specific Homer residues for ligand binding. These results establish a molecular basis for understanding the biological properties of Homer-ligand complexes.     

Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.