平成22・23年度「独創性のある生命科学研究」個別研究課題 3）精子における活性酸素消去機構の解析

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Tubular Injury in a Rat Model of Type 2 Diabetes Is Prevented by Metformin: A Possible Role of HIF-1α Expression and Oxygen Metabolism

http://dx.doi.org/10.2337/db10-0655OBJECTIVE : Chronic hypoxia has been recognized as a key regulator in renal tubulointerstitial fibrosis, as seen in diabetic nephropathy, which is associated with the activation of hypoxia-inducible factor (HIF)-1α. We assess here the effects of the biguanide, metformin, on the expression of HIF-1α in diabetic nephropathy using renal proximal tubular cells and type 2 diabetic rats. RESEARCH DESIGN AND METHODS : We explored the effects of metformin on the expression of HIF-1α using human renal proximal tubular epithelial cells (HRPTECs). Male Zucker diabetic fatty (ZDF; Gmi-fa/fa) rats were treated from 9 to 39 weeks with metformin (250 mg/kg^/day^) or insulin. RESULTS : Metformin inhibited hypoxia-induced HIF-1α accumulation and the expression of HIF-1–targeted genes in HRPTECs. Although metformin activated the downstream pathways of AMP-activated protein kinase (AMPK), neither the AMPK activator, AICAR, nor the mTOR inhibitor, rapamycin, suppressed hypoxia-induced HIF-1α expression. In addition, knockdown of AMPK-α did not abolish the inhibitory effects of metformin on HIF-1α expression. The proteasome inhibitor, MG-132, completely eradicated the suppression of hypoxia-induced HIF-1α accumulation by metformin. The inhibitors of mitochondrial respiration similarly suppressed hypoxia-induced HIF-1α expression. Metformin significantly decreased ATP production and oxygen consumption rates, which subsequently led to increased cellular oxygen tension. Finally, metformin, but not insulin, attenuated tubular HIF-1α expression and pimonidazole staining and ameliorated tubular injury in ZDF rats. CONCLUSIONS : Our data suggest that hypoxia-induced HIF-1α accumulation in diabetic nephropathy could be suppressed by the antidiabetes drug, metformin, through the repression of oxygen consumption

Measurement of melatonin in body fluids: Standards, protocols and procedures

Abstract: The circadian rhythm of melatonin in saliva or plasma, or of the melatonin metabolite 6‐ sulphatoxymelatonin in urine, is a defining feature of suprachiasmatic nucleus function, the endogenous oscillatory pacemaker. These measurements are useful to evaluate problems related to the onset or offset of sleep and for assessing phase delays or advances of rhythms in entrained individuals. Additionally, they have become an important tool for psychiatric diagnosis, its use being recommended for phase typing in patients suffering from sleep and mood disorders. Thus, the development of sensitive and selective methods for the precise detection of melatonin in tissues and fluids of animals emerges as necessary. Due to its low concentration and the co‐existence of many other endogenous compounds in blood, the determination of melatonin has been an analytical challenge. This review discusses current methodologies employed for detection and quantification of melatonin in biological fluids and tissues

Separation and assay methods for melatonin and its precursors

ELSEVIER, Harumi, T; Matsushima, S, JOURNAL OF CHROMATOGRAPHY. B, BIOMEDICAL AND APPLICATIONS, 747(1-2), 95-110, 2000. authorMelatonin is an indoleamine hormone that is synthesized from tryptophan via 5-hydroxytryptophan, serotonin and N-acetylserotonin in the vertebrate pineal gland. Many chromatographic and non-chromatographic techniques have been developed and improved for the determination and measurement of melatonin and its related indoleamines. At present, gas chromatography with mass spectrometry and reversed-phase high performance liquid chromatography with fluorescence or electrochemical detection are widely used for indoleamine determinations in the pineal gland. This review will deal with methods for the separation and determination of the melatonin and its related indoleamines

精子における活性酸素消去機構の解析

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Characterization of Sea Urchin Sperm Membrane Proteins which Interact with a Major Acrosome Reaction-Inducing Substance, Fucose Sulfate Glycoconjugate

Intact sea urchin spermatozoa were successfully biotinylated with NHS-LC-Biotin and the biotinylated spermatozoa retained the viability. Analysis of the membrane prepared from the biotinylated spermatozoa of the sea urchin Hemicentrotus pulcherrimus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that several proteins such as wheat-germ agglutinin (WGA)-binding protein (220 kDa), guanylyl cyclase (131 kDa), sperm-activating peptide-I (SAP-I)-crosslinking protein (71 kDa), GPI-anchored protein (63 kDa) and functionally unknown proteins (50 kDa and 30 kDa) were specifically biotinylated. Experiments using spermatozoa of sea urchins, Anthocidaris crassispina and Clypeasfer japonicus showed that several proteins similar to those of H. pulcherrimus spermatozoa were also labeled with NHS-LC-Biotin. Fucose sulfate glycoconjugate (FSG) isolated from the jelly coat of H. pulcherrimus was mixed with solubilized biotinylated sperm membrane proteins of H. pulcherrimus, A. crassispina or C. japonicus and then subjected to gel filtration chromatography on a Sepharose 2B column, indicating that only two biotinylated H. pulcherrimus sperm proteins were coeluted with H. pulcherrimus FSG

Expression of Membrane-Bound and Soluble Guanylyl Cyclase mRNAs in Embryonic and Adult Retina of the Medaka Fish Oryzias latipes

Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OIGC3, OIGC4, OIGC5, and OIGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OIGCS-α1, OIGCS-α2, and OIGCS-β1, neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OIGC3 and OIGC5 mRNAs were expressed in the proximal part and the OIGC4 and OIGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OIGCS-α2 and OIGCS-β1) of two soluble GC subunits (α2 and β1) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GCα1 subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (α2/β1 heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas