新規カルボニル還元酵素およびマラリア原虫由来酵素の構造生物学的研究

筑波大学University of Tsukuba博士（生物科学）Doctor of Philosophy in Biological Science2017thesi

Denaturation, Renaturation and Other Structural Studies on Phosphoglucose Isomerases

Structural properties of phosphoglucose isomerases isolated from a variety of species have been compared by peptide fingerprinting, predicted amino acid sequence homologies and by denaturation and renaturation studies. The enzymes are more readily denatured in guanidinium chloride than in urea, and the isomerase isolated from yeast is more stable toward acid pH than the rabbit muscle enzyme. The rates of guanidinium chloride-induced denaturation are markedly increased by ionic strength and decreased by substrates, competitive inhibitors or glycerol. The enzyme can be renatured, but only in the presence of glycerol. The renaturation process is dependent on protein concentration and temperature and provides a method for the formation of mixed species heterodimers

Human Phosphoglucose Isomerase: Isolation and Characterization of Wild Type and the Singh Allozyme

A procedure was developed for the rapid isolation of human phosphoglucose isomerase by substrate-induced elution from cellulose phosphate. The high degree of selectivity of the elution provided homogenous enzyme from erythrocytes after a purification of approximately 30,000-fold with a recovery of approximately 70%. The enzyme was also isolated from other human tissues by a similar procedure

Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity

Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells

Indole-3-acetic acid regulates the central metabolic pathways in Escherichia coli.

The physiological changes induced by indoleacetic acid (IAA) treatment were investigated in the totally sequenced Escherichia coli K-12 MG1655. DNA macroarrays were used to measure the mRNA levels for all the 4290 E. coli protein-coding genes; 50 genes (1-1 %) exhibited significantly different expression profiles. In particular, genes involved in the tricarboxylic acid cycle, the glyoxylate shunt and amino acid biosynthesis (leucine, isoleucine, valine and proline) were up-regulated, whereas the fermentative adhE gene was down-regulated. To confirm the indications obtained from the macroarray analysis the activity of 34 enzymes involved in central metabolism was measured; this showed an activation of the tricarboxylic acid cycle and the glyoxylate shunt. The malic enzyme, involved in the production of pyruvate, and pyruvate dehydrogenase, required for the channelling of pyruvate into acetyl-CoA, were also induced in IAA-treated cells. Moreover, it was shown that the enhanced production of acetyl-CoA and the decrease of NADH/NAD+ ratio are connected with the molecular process of the IAA response. The results demonstrate that IAA treatment is a stimulus capable of inducing changes in gene expression, enzyme activity and metabolite level involved in central metabolic pathways in E. col

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.Ministerio de Ciencia e Innovación AGL2011-2318

Mechanisms of activation of glycogenolysis during development of malignant hyperthermia in swine

The syndrome of Malignant Hyperpyrexia in man follows administration of certain general anaesthetic agents, and, although rare, is fatal in 70% of cases (EDITORIAL, 1968). Following exposure to the anaesthetic, there is, in most instances of susceptible individuals, a rapid rise in body temperature, usually within a period of 10 minutes, often accompanied by muscular rigidity of the limbs~ Sometimes hyperthermia has.been delayed for hours and muscular rigidity not pronounced. The temperature reached maybe 43°C (115°F) or even somewhat above this. Halothane, CF3CHBrCl, a ha../o/~nated hydrocarbon, is thought to be responsible for most cases(WILSON, NICHOLS, DENT and ALLEN, 1966). Succinyl choline lfH2COOCH2CH2*(cH3 )~ 201_~H2COOCH2CH2~(cH3 )3 a skeletal muscle relaxant employed during anaesthesia, has also been implicated (BRITT and KALOW, 1970; HARRISON, 1971)