Pathways of ultraviolet mutability in Saccharomyces Cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance.

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr⁺ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yeild a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7, and α umr7 were equally sensitive to an intermediate degree. Incubation at 30°C of wildtype cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEDP growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which had moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

ADMA induces monocyte adhesion via activation of chemokine receptors in cultured THP-1 cells

Asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, is also an important inflammatory factor contributing to the development of atherosclerosis (AS). The present study was to test the effect of ADMA on angiotensin (Ang) II-induced monocytic adhesion. Human monocytoid cells (THP-1) or isolated peripheral blood monocyte cells (PBMCs) were incubated with Ang II (10⁻⁶ M) or exogenous ADMA (30 μM) for 4 or 24 h in the absence or presence of losartan or antioxidant PDTC. In cultured THP-1 cells, Ang II (10⁻⁶ M) for 24 h elevated the level of ADMA in the medium, upregulated the protein expression of protein arginine methyltransferase (PRMT) and decreased the activity of dimethylarginine dimethylaminohydrolase (DDAH). Both of Ang II and ADMA increased monocytic adhesion to human umbilical vein endothelial cells (HUVECs), elevated the levels of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8 and tumor necrosis factor (TNF)-α and upregulated CCR₂ and CXCR₂ mRNA expression, concomitantly with increase in reactive oxygen species (ROS) generation and activation of nuclear factor (NF)-κB. Pretreatment with losartan (10 μM) or PDTC (10 μM) abolished the effects mediated by Ang II or ADMA. In isolated PBMCs from healthy individuals, ADMA upregulated the expression of CXCR₂ mRNA, which was attenuated by losartan (10 μM), however, ADMA had no effect on surface protein expression of CCR₂. The present results suggest that ADMA may be involved in monocytic adhesion induced by Ang II via activation of chemokine receptors by ROS/NF-κB pathway.     

Protein arginine methylation regulates insulin signaling in L6 skeletal muscle cells

Protein N-arginine methyltransferase (PRMT)1 catalyzes arginine methylation in a variety of substrates, although the potential role of PRMT1 in insulin action has not been defined. We therefore investigated the effect of PRMT1-mediated methylation on insulin signaling and glucose uptake in skeletal L6 myotubes. Exposure of L6 myotubes to insulin rapidly induced translocation of PRMT1 and increased its catalytic activity in membrane fraction. Several proteins in the membrane fraction were arginine-methylated after insulin treatment, which were inhibited by pretreatment with an inhibitor of methyltransferase, 5′-deoxy-5′-(methylthio)adenosine (MTA), or a small interfering RNA against PRMT1 (PRMT1-siRNA). Inhibition of arginine methylation with MTA or PRMT1-siRNA diminished later phase of insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) β and IRS-1, association of IRS-1 with p85α subunit of PI3-K, and glucose uptake. Our results suggest that PRMT1-mediated methylation serves as a positive modulator of IR/IRS-1/PI3-K pathway and subsequent glucose uptake in skeletal muscle cells.     

Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.     

arcA基因提高大肠杆菌对有机溶剂的耐受性

【目的】将来源于恶臭假单胞菌(Pseudomonas putida JUCT1)的基因arc A(编码精氨酸脱亚胺酶)整合到Escherichia coli JM109(DE3)基因组中,以提高该菌对有机溶剂的耐受性。【方法】以P.putida JUCT1的基因组为模板扩增基因arc A,并与p ET-20b(+)连接后导入E.coli JM109(DE3)中,验证该基因提高E.coli JM109(DE3)对有机溶剂的耐受性。利用Red同源重组的方法将arc A整合到E.coli JM109(DE3)基因组中。【结果】E.coli JM109(DE3)/p ET-20b(+)-arc A在添加了2.0%(体积比)环己烷、0.1%(体积比)甲苯、4.0%(体积比)萘烷和0.1%(体积比)丁醇的培养基中培养8 h后,其OD660由初始的0.2分别上升到0.8、0.9、1.8和1.3。将arc A成功整合到E.coli JM109(DE3)基因组中,获得了具有较好遗传稳定性的溶剂耐受E.coli JM109(DE3)宿主菌株。【结论】外源基因arc A能提高大肠杆菌菌株的有机溶剂耐受性,为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。     

Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies

Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.     

Analysis of Evolutionarily Independent Protein-RNA Complexes Yields a Criterion to Evaluate the Relevance of Prebiotic Scenarios

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改良法建立妊娠期高血压疾病大鼠模型

目的寻找理想的妊娠期高血压疾病动物模型。方法60只雌性Wistar大鼠分为六组,分别为正常妊娠组（A组）、左旋亚硝酸精氨酸甲酯（L-NAME）腹腔注射组（B组）、生理盐水注射组（C组）、假手术组（D组）、双侧子宫动脉结扎术组（E组）以及L-NAME腹腔注射联合腹主动脉缩窄术组（F组）,对大鼠血压、尿蛋白值以及胎鼠、胎盘、胎鼠头的重量进行监测并观察肾脏、胎盘的病理改变。结果F组孕鼠于孕13d即出现了蛋白尿、血压升高,较B组及E组出现时间早（P〈0.05）,其胎鼠体重、胎头重量下降较各组更明显（P〈0.05）,肾小球小动脉管壁增厚、管腔狭窄;胎盘出现血管间膜增厚、纤维蛋白沉积等妊娠高血压疾病的典型病理改变。结论L-NAME腹腔注射联合腹主动脉缩窄术是更为理想的建立妊娠高血压疾病动物模型的方法。