Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.     

Effects of solubilisation on some properties of the membrane-bound respiratory enzyme D-amino acid dehydrogenase of Escherichia coli

Solubilisation, delipidation and partial purification of the membrane-bound enzyme D-amino acid dehydrogenase of Escherichia coli K12 produced significant changes in several of its properties. Solubilised enzyme showed a broader substrate specificity, increased affinity for at least three substrates, and a lower pH optimum with D-alanine as substrate. Solubilised enzyme was more heat-labile than native enzyme, particularly at 37 degrees C, and re-binding to envelope preparations restored protection against heat denaturation. Activity of delipidated enzyme could be increased by addition of pure phospholipids. Native enzyme showed biphasic Arrhenius kinetics associated with phase changes of membrane lipids.     

Relationship of alpha MSH-specific neurons to the arcuate opiocortin neuronal system as determined by dual antigen immunocytochemical procedures

The cross-immunoreactivity, topography, and fiber projections of the alpha MSH-immunoreactive specific neurons in the forebrain of the rat appear to be distinctly different from that of the neurons in the hypothalamic arcuate opiocortin system. The cell bodies, immunoreactive only to -MSH, have a specific pattern of distribution in the dorsal and lateral hypothalamic regions from the level of the retrochiasmatic region to the premammillary area of the posterior hypothalamus. Immunoreactive fibers of these cells appear to extend into regions of the cerebral cortex and hippocampus. An antomical relationship between the immunostained fibers and/or terminals of the arcuate opiocortin pool of neurons and the -MSH-immunoreactive perikarya is described utilizing the ABC (Avidin-Biotin-Peroxidase Complex) and ABC-GO (Glucose Oxidase) or glucose oxidase-antiglucose oxidase complex methods of immunocytochemistry in which two tissue antigens with contrasting colors are demonstrated in the same tissue section.     

Effect of triethyltin on Escherichia coli K-12.

Triethyltin (TET) stimulated the basal respiration of Escherichia coli K-12 membrane vesicles in chloride (Cl-) medium but it had little effect on respiration in sulphate (SO4(2-)) medium. Since this uncoupling activity was Cl- dependent it was attributed to the Cl-/hydroxide (OH-) exchange reaction known to be mediated by TET [1,2]. TET inhibited the oxidation of succinate by intact E. coli in both Cl- and SO4(2-) medium, but at the same concentration of TET, inhibition was always more extensive in Cl- than SO4(2-) medium. In Cl- medium uncoupling in membrane vesicles and inhibition of succinate oxidation in intact bacteria occurred over the same concentration range and it appeared that the same mechanism, i.e. Cl-/OH- exchange, was responsible for both effects. Inhibition of succinate oxidation in SO4(2-) medium was not substantial until the concentration of TET was greater than 10(-5) M. Although the nature of this inhibition could not be determined by experiments with membrane vesicles indirect evidence from growth experiments indicated that it was due to impairment of oxidative phosphorylation. The relationship between these biochemical findings and the bacteriocidal action of TET was examined by using various concentrations of anion and substrate in the growth medium. Growth was inhibited in media containing either Cl- or SO4(2-) as the main anion but at a particular concentration of TET, inhibition was greater in Cl- medium. Growth was also inhibited to a greater extent in succinate than glucose medium. Furthermore in either Cl- or SO4(2-) glucose medium, lactic acid production increased as the concentration of TET was increased. These findings imply that the bacteriocidal action of TET is related to its effect(s) on oxidative phosphorylation.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

2021-01期目录

为研究团头鲂(Megalobrama amblycephala) 5-甲基胞嘧啶(5-methylcytosine, 5mC)羟基化酶TET1(Ten eleven translocation 1)基因的功能及其表达特性, 采用整胚原位杂交、qRT-PCR技术进行了胚胎、组织表达分析及其在急性低氧胁迫下的基因响应研究。序列分析结果表明, 团头鲂TET1基因全长为5526 bp, 编码1841个氨基酸。qRT-PCR结果表明, 团头鲂TET1广泛表达于各个组织中, 并在脑组织中高度表达。在胚胎发育过程中, TET1基因从受精卵开始就有表达, 并在受精后20—44h (20—44 hpf)都维持在一个较高水平。原位杂交结果表明, TET1基因在12 hpf信号相对微弱, 在24和36 hpf信号逐渐增强, 并且都集中在头部表达。通过qRT-PCR检测急性缺氧处理TET1的表达量, 结果表明, TET1基因在鳃和脾等组织中表达显著升高(P<0.01), 在脑、皮肤、眼和肾脏中表达显著降低(P<0.01)。在胚胎中, TET1基因相对表达量明显高于对照组, 尤其在24 hpf低氧处理组的表达量显著地高于对照组(P<0.001)。结果表明TET1基因在低氧应答反应中发挥着重要的作用。该研究结果为TET1基因在低氧响应及功能的保守与分化方面提供了新的视角。     

1-Methyl-3-nitropyridine: An Efficient Oxidant of NADH in Non-enzymatic and Enzyme-mediated Processes

It is shown that NADH can be effectively oxidized by 1-methyl-3-nitropyridine in non-enzymatic and enzyme-mediated processes. Mechanistic issues of these reactions are discussed. These processes seem to contribute to the observed cytotoxicity of 1-methyl-3-nitropyridine. A key role of 1-methyl-3-nitropyridinyl radical formed in the enzyme-mediated processes is emphasized.