Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 degrees C) and all other methanongens (84-86 degrees C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.     

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.     

微生物亚硝酸盐还原酶的研究进展

亚硝酸盐还原酶(Nitrite reductase,简称NiR,EC1.7.2.1)是催化亚硝酸盐(Nitrite,简称NIT)还原的一类酶,可降解NIT为NO或NH3,是自然界氮循环过程的关键酶。本文详细阐述亚硝酸盐还原酶的分类、结构特点、催化机制以及现阶段的应用领域,为深入研究亚硝酸还原酶提供参考。     

Characterization of Desulfomicrobium salsuginis sp. nov. and Desulfomicrobium aestuarii sp. nov., two new sulfate-reducing bacteria isolated from the Adour estuary (French Atlantic coast) with specific mercury methylation potentials

Three strains of sulfate-reducing bacteria (ADR21, ADR26 and ADR28) were isolated from Adour estuary sediments (French South Atlantic coast). Cells of these isolates were rod-shaped, motile and stained Gram-negative. The 16S rRNA and dsrAB genes sequence analyses indicated that these three strains belonged to the genus Desulfomicrobium within the delta Proteobacteria, with Desulfomicrobium escambiense strain DSM10707^T as their closest relative. According to phenotypic characteristics, strains ADR21 and ADR28 could be considered as members of the same species. The relatedness values, based on DNA–DNA hybridization studies, between strains ADR21/DSM10707^T, ADR26/DSM10707^T and ADR21/ADR26 ranged between 30.6–40.8%, 45.2–43.0% and 19.0–26.4%, respectively. Strains ADR21 and ADR28 grew well on lactate, fumarate, malate, formate, ethanol and H₂/acetate in the presence of sulfate as an electron acceptor. Thiosulfate, nitrate, fumarate and DMSO were alternative electron acceptors. Malate was well fermented but pyruvate and fumarate only poorly. Strain ADR26 could not grow on ethanol or fumarate and was unable to use DMSO or fumarate as electron acceptors. The three new strains exhibited differences compared to the type strain of D. escambiense, such as temperature optima, substrate utilization and mercury methylation capacities. On the basis of both genetic and phenotypic evidences, strain ADR21 is proposed as the type strain of the species Desulfomicrobium salsuginis sp. nov., and strain ADR26 as the type strain of the species Desulfomicrobium aestuarii sp. nov.     

不同培养基和三种金属离子对桦褐孔菌培养菌丝体的羊毛甾醇和麦角甾醇积累的影响

Lanosterol and ergosterol are the active principles with potential pharmacological activities in Inonotus obliquus. However, the two sterols are less accumulated in cultured mycelia of the fungus. In this study, different carbon and nitrogen sources and pH levels together with three metal ions were assayed for their effects on accumulation of the two sterols in the fungus. Among the tested media the growth medium consisting of glucose (1.5%), rice powder (0.5%), yeast extract (0.4%), wheat bran (0.1%), KH2PO4 (0.01%) and MgSO4?7H2O (0.05%) with pH level at 6.5 yielded a maximum production of the two sterols, which can further be increased following the treatment of Ag+, Cu2+ and Ca2+. Supplementing Ag+ at concentrations of 0.28 and 0.35?mol partially inhibited ergosterol biosynthesis, leading to an enhanced accumulation of lanosterol, the presence of intermediates of ergosterol biosynthetic pathway and a reduced accumulation of ergosterol in cultured mycelia of I. obliquus.     

Amelioration of cadmium-induced cardiac impairment by taurine

The present study has been designed to investigate the protective role of taurine (2-aminoethanesulfonic acid), a sulfur containing conditionally essential amino acid, against cadmium-induced cardiac dysfunction in mice. Cadmium chloride (CdCl(2)) was used as the source of cadmium and it was administered orally at a dose of 4mg/kg body weight for 6 days. Cadmium exposure caused significant accumulation of the cadmium and iron in mice hearts tissue. Levels of serum specific markers related to cardiac impairments, e.g. total cholesterol, HDL cholesterol and triglyceride were altered due to cadmium toxicity. Reduction in the activities of antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD) have been observed in cadmium exposed mice. Cadmium intoxication also decreased the cardiac glutathione (GSH) and total thiols contents and increased the levels of oxidized glutathione (GSSG), lipid peroxidation end products, protein carbonyl content and the extent of DNA fragmentation. Oral administration of taurine at a dose of 100mg/kg body weight for 5 days, however, prevented all the toxin-induced oxidative impairments mentioned above. "Ferric Reducing/Antioxidant Power (FRAP) assay" showed that taurine could protect the cardiac tissue by preventing cadmium-induced reduction of the intracellular antioxidant power. Histological examination of cardiac segments also supported the beneficial role of taurine against cadmium-induced damages in the murine hearts. Effect of a well established antioxidant, vitamin C has been included in the study as a positive control. Combining all, results suggest that taurine attenuates cadmium-induced impairment in mice hearts.     

醛糖还原酶基因5′调控区新的点突变对基因表达调控的影响

为了进一步研究醛糖还原酶 (AR)基因 5′调控区存在的可引起蛋白质表达发生改变的遗传变异及其对糖尿病并发症的影响 ,应用PCR SSCP对中国人 2型糖尿病患者的AR基因 5′调控区进行筛选 ,在两名糖尿病患者中发现一新点突变C- 1 6 7→A ,使AR基因 5′调控区产生一个新的CCAAT盒。含点突变的两名患者尽管患病多年 ,长期处于高血糖状态 ,但无糖尿病并发症发生。而且他们的红细胞中AR活性都很低 ,处于无视网膜病变患者组的下限范围。将含野生型和点突变DNA片段分别克隆至氯霉素乙酰转移酶报告基因载体 (pCAT) ,检测CAT的活性确定野生型和突变型序列的转录活性。同时进行凝胶滞留试验以观测DNA与蛋白质的相互作用。结果显示 :含C- 1 6 7→A的启动子相对转录活性 (5 .7% )明显低于野生型(15 .7% )。凝胶滞留试验中 ,突变序列迁移速率较野生型慢。以上结果说明 ,AR基因 5′调控区C- 1 6 7→A点突变干扰了启动子区顺式作用元件与反式作用因子的结合 ,导致AR基因转录活性降低 ,使患者组织中AR活性下降 ,从而阻止或减缓 2型糖尿病视网膜病变发生发展。     

Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus

Abstract The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus , was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA ) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order.     

The activity and stability of wheat nitrate reductase in vitro

The activity and decay characteristics of nitrate reductase from wheat (Triticum aestivum) were studied in crude, partially-purified and highly-purified preparations. The decay of nitrate reductase activity in crude extracts was due to spontaneous dissociation of the enzyme and to the effects of two decay factors, one present in the 0–30% and the other in the 50–70% saturated (NH₄)₂SO₄ fraction of a crude extract. Low rates of factor-mediated NR decay in vitro were associated with high levels of NR activity in vivo.     

Hexokinase receptors: Preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude mitochondrial fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude mitochondrial fraction, a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to non-mitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.