Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN.

Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot- (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot- defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Incorporation of Molecular Oxygen and Water during Enzymatic Oxidation of Cyanide by Pseudomonas fluorescens NCIMB 11764

Cell extracts (high-speed [150,000 x g] supernatants) from Pseudomonas fluorescens NCIMB 11764 catalyzed the oxidation of cyanide to CO(inf2) (and NH(inf3)). Conversion was both oxygen and NADH dependent, with 1 mol of each being consumed per mol of cyanide degraded. Analysis of (sup13)CO(inf2) by mass spectrometry indicated that one atom each of isotopically labelled oxygen 18 from molecular oxygen and water were incorporated during enzymatic conversion. The results confirm earlier reports of oxygenase-mediated cyanide conversion in this organism. A reaction pathway for cyanide oxidation involving initial monooxygenation followed by hydrolysis of a hypothetical oxygenated intermediate to CO(inf2) (and NH(inf3)) is proposed.     

On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc.     

以淀粉珠为载体的亲和层析法分离纯化高温α淀粉酶

 以淀粉珠为载体的亲和层析法分离纯化高温α淀粉酶张学忠,宋伦,王群,吴晓霞,唐锌进（吉林大学酶工程国家重点实验室,长春１３００２３;南京师范大学生物系,南京２１００２４）金凤燮等人从酒曲中筛选出高产热稳定α淀粉酶的菌株,命名为Ｂａｃｉｌｌｕｓｓｐ－ＪＦ...     

汉防己甲素及克矽平对矽肺大鼠Ⅰ型和Ⅲ型胶原mRNA的影响

汉防己甲素（汉甲）及克矽平（Ｐｏｌｙｖｉｎｙｌｐｙｒｉｄｉｎｅ－Ｎ－Ｏｘｉｄｅ,ＰＶＮＯ）是目前较为有效的抑制矽肺纤维化的药物。本文研究了其对胶原ｍＲＮＡ水平的影响．斑点杂交实验表明大鼠接尘６０天和１２０天后α１（Ⅰ）及α１（Ⅲ）ｍＲＮＡ水平明显上升,经汉甲或克矽平治疗１个月或３个月后,胶原ｍＲＮＡ水平明显下降。原位杂交结果表明胶原ｍＲ－ＮＡ银颗粒与细胞性结节和增厚的肺泡壁的成纤维细胞分布重合。汉甲或克矽平治疗后银颗粒数下降。提示汉甲及克矽平对矽肺进程中的胶原基因表达增强有抑制作用。     

玉米根细胞中对钒酸盐敏感的H~＋－泵的研究

用奎吖咽（ｑｕｉｎａｃｒｉｎｅ）作荧光指标剂,测定玉米（ＺｅａｍａｙｓＬ．）根尖微粒体（ＭＩＣ）膜囊泡的Ｈ~＋－泵活性,结果表明１ｍｍｏｌ／ＬＮａＮ_３仅抑制该泵活性约８％,而０．８ｍｍｏｌ／Ｌ钒酸盐（Ｖａｎ）则可抑制其活性达８０％,说明ＭＩＣ制剂中Ｈ~＋－泵活性主要由质膜（ＰＭ）Ｈ~＋－ＡＴＰａｓｅ产生。此泵活性严格需要Ｍｇ~（２＋）,二价阳离子作用大小的顺序为Ｍｇ~（２＋）＞Ｍｎ~（２＋）＞Ｚｎ~（２＋）＞Ｃａ~（２＋）＝０;阴离子作用大小的１顺序为Ｂｒ~－＞Ｃｌ~－＞ＮＯ_３~－＞ＳＯ_４~（２－）,并初步证实当质膜同侧发生电子传递时,没有跨膜Ｈ~＋梯度（△μＨ~＋）生成。