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Biomedical subjects

M Arisawa

Publications and source records attributed to M Arisawa.

At least 19 recordsLinked to original sources
PubMed2001

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma.

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC(50) of 10.7 microM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[(14)C]arginine to L-[(14)C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.

Animals↗
PubMed2001

Regulation of catalytic activity of a multifunctional polyketide biosynthetic enzyme, 6-hydroxymellein synthase, by interaction between NADPH and phenylglyoxal-sensitive amino acid residue at the reaction center.

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification of the enzyme protein with phenylglyoxal. Appreciable differences in K(m) values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalytic activity of the NADPH-associated synthase was enhanced by the addition of free CoA, the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme. A marked deuterium isotope effect in the catalytic reaction of the native synthase-NADPH complex was appreciably decreased in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2'-position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.

Acyltransferases↗
PubMed2001

Discovery of novel aldose reductase inhibitors using a protein structure-based approach: 3D-database search followed by design and synthesis.

Aldose reductase (AR) has been implicated in the etiology of diabetic complications. Due to the limited number of currently available drugs for the treatment of diabetic complications, we have carried out structure-based drug design and synthesis in an attempt to find new types of AR inhibitors. With the ADAM&EVE program, a three-dimensional database (ACD3D) was searched using the ligand binding site of the AR crystal structure. Out of 179 compounds selected through this search followed by visual inspection, 36 compounds were purchased and subjected to a biological assay. Ten compounds showed more than 40% inhibition of AR at a 15 microg/mL concentration. In a subsequent lead optimization, a series of analogues of the most active compound were synthesized based on the docking mode derived by ADAM&EVE. Many of these congeners exhibited higher activities compared to the mother compound. Indeed, the most potent, synthesized compound showed an approximately 20-fold increase in inhibitory activity (IC(50) = 0.21 vs 4.3 microM). Furthermore, a hydrophobic subsite was newly inferred, which would be useful for the design of inhibitors with improved affinity for AR.

Aldehyde Reductase↗
PubMed2001

Rhodium-catalyzed Beckmann rearrangement.

[figure: see text] Beckmann rearrangement of oxime is catalyzed by [RhCl(cod)]2, trifluoromethanesulfonic acid, and tris(p-tolyl)phosphine in refluxing dichloroethane, giving the corresponding amide in good yield. Product/acid ratios of 10:20 can be attained in the reaction of benzophenone oximes.

Journal Article↗
PubMed2001

Hypervalent iodine(III)-induced intramolecular cyclization of alpha-(aryl)alkyl-beta-dicarbonyl compounds: a convenient synthesis of benzannulated and spirobenzannulated compounds.

A novel hypervalent iodine(III)-induced direct intramolecular cyclization of alpha-(aryl)alkyl-beta-dicarbonyl compounds has been described. Both meta- and para-substituted phenol ether derivatives containing acyclic or cyclic 1,3-dicarbonyl moieties at the side chain undergo this reaction in a facile manner. The reactions afford benzannulated and spirobenzannulated compounds that are of biological importance. The reaction is found to be general, mild, and high yielding. The mechanism of the reaction has been shown to involve a cation radical intermediate.

Journal Article↗
PubMed2001

Dihydroxynortropane alkaloids from calystegine-producing plants.

Three dihydroxynortropanes, 2alpha,7beta-dihydroxynortropane, 2alpha,3beta-dihydroxynortropane, and 3alpha,7beta-dihydroxynortropane, were isolated from calystegine-producing plants in the families Convolvulaceae and Solanaceae. 2alpha,7beta-Dihydroxynortropane was isolated from six species in the Convolvulaceae whereas only Calystegia soldanella contained it and 2alpha,3beta-dihydroxynortropane. Although neither of these were detectable in three species tested in the Solanaceae, 3alpha,7beta-dihydroxynortropane was, however, isolated from Duboisia leichhardtii.

Alkaloids↗
PubMed2001

Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells.

Biosynthetic activity of carrot phytoalexin 6-methoxymellen was induced in cell suspension culture by the treatment with oligogalacturonide elicitor; however, the elicitor-induced activity appreciably reduced in the presence of suramin, a potent inhibitor of GTP-binding proteins. In contrast, addition of G-protein activators, such as mastoparan or GTP-gamma-S, to carrot cell culture triggered 6-methoxymellein production even in the absence of uronide elicitor. An appreciable GTPase activity was found in purified plasma membrane of cultured carrot cells, and the hydrolytic activity was significantly increased by the addition of elicitor. Carrot plasma membrane was capable of associating with GTP-gamma-S, and the binding ability was markedly increased in the presence of elicitor. However, the binding activity markedly decreased when the membrane preparation was pre-incubated with GTP but not with ATP. These observations strongly suggest that a certain GTP-binding protein located at plasma membrane of cultured carrot cells plays an important role in the oligogalacturonide elicitor-induced 6-methoxymellein production.

Journal Article↗
PubMed2001

Mechanisms of fluconazole resistance in Candida albicans isolates from Japanese AIDS patients.

Four Candida albicans isolates, TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166, with reduced fluconazole susceptibility were obtained from three AIDS patients in Japan, and the mechanisms of their drug resistance were studied. All isolates showed lower levels of intracellular accumulation of fluconazole than ATCC 10231, a susceptible control strain of C. albicans. Increased amounts of CDR1 and CDR2 mRNA encoding putative ATP binding cassette (ABC) transporters were associated with the azole resistance of all TIMM isolates, apart from TIMM 3164. In addition, increased Cdr1p levels were immunodetected in the cell membrane fractions of all the TIMM strains except for TIMM 3164. Gene amplification was not responsible for CDR1 overexpression and there were no significant differences in the mRNA levels of CDR3 or CDR4 (ABC transporters) in the azole-susceptible and -resistant cells. CaMDR1 (a major facilitator superfamily) gene expression was not observed in any of the resistant isolates or the control strain. These results suggest that energy-dependent drug efflux associated with increased expression of CDR1 and CDR2 is involved in the fluconazole resistance mechanisms in two of the four isolates, TIMM 3165 and TIMM 3166. TIMM 3164 demonstrated energy-dependent drug efflux without overexpression of CDR1-4 or CaMDR1, indicating that some other pump may be operating. Despite showing low levels of drug efflux and overexpression of CDR1 and CDR2, efflux in TIMM 3163 was not energy dependent, suggesting that the expressed Cdr1p non-functional Cdr1p and that other resistance mechanisms may operate in this strain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

In vitro and in vivo effects of 14alpha-demethylase (ERG11) depletion in Candida glabrata.

Sterol 14alpha-demethylase (ERG11) is the target enzyme of azole antifungals that are widely used for the treatment of fungal infections. Candida glabrata is known to be less susceptible to fluconazole than most Candida albicans strains, and the incidence of C. glabrata infection has been increasing mostly in conjunction with the use of azole antifungals. Recently, it has been reported that C. glabrata can rescue the defect of ergosterol biosynthesis by incorporating cholesterol from serum. To explore the effect of inactivating Erg11p in C. glabrata, we generated mutant strains in which the ERG11 gene was placed under the control of tetracycline-regulatable promoters. In these mutants, expression of the ERG11 gene can be repressed by doxycycline (DOX). All mutants showed a growth defect in the presence of DOX. The numbers of CFU of the mutants were lowered by only 1/10 with DOX treatment. In these mutants, accumulation of 4,14-dimethylzymosterol, which differs from an accumulated abnormal sterol detected in C. albicans and Saccharomyces cerevisiae treated with fluconazole, was observed by DOX treatment. Although such phenotypes were also observed in serum-containing media by DOX treatment, they were alleviated. Furthermore, the mutant could grow in DOX-treated mice without a severe reduction in the number of cells. Thus, depleting the expression of the ERG11 gene lowered the number of CFU by only 1/10 due to the accumulation of 4,14-demethylzymosterol in vitro, and it did not result in the defective growth of fungal cells in mice. These results suggested that Erg11p is not an ideal target molecule of antifungals for C. glabrata.

Anti-Bacterial Agents↗
PubMed2000

Role of reducing co-factors in catalytic reactions of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells.

6-Hydroxymellein (6HM) synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, is capable of catalyzing the acyl-CoA condensation and the ketoreduction in the presence of the nucleotide reducing co-factors. Although free CoA at high concentrations functioned as the activator of the NADPH-dependent 6HM formation, the compound exhibited an appreciable inhibitory activity toward the reaction mediated by NADH. CoA showed a potent inhibitory activity against substrate entry into the reaction center of the NADH-associated enzyme while, in the presence of NADPH, the compound slightly inhibited the formation of the acylated enzyme. The catalytic rate of the synthase was appreciably decreased when NADPH was replaced by the deuterium-labeled compound, however, the kH/kD value was markedly reduced if NADH and [D]NADH were employed as the reducing co-factors. These results suggest that the phosphate group attached to 2'-position of the adenosyl moiety of NADPH associated with the ketoreducing domain of 6HM synthase plays an important role in the regulation of the enzyme activity.

Journal Article↗
PubMed2000

Identification of a novel inhibitor specific to the fungal chitin synthase. Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans.

As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K(i) value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC(50) value of 0.27 microm. In the presence of RO-09-3143, the C. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2Delta null mutants but not in the cachs3Delta null mutants. Thus, it appears that in C. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.

Aminoglycosides↗
PubMed2000

Unusual arrangement of catalytic domains in head-to-tail associated homodimer of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme.

6-Hydroxymellein synthase, a multifunctional polyketide synthetic enzyme in carrot, is organized as a homodimer, and the activity of the synthase was appreciably inhibited upon the specific alkylation of cysteine- and cysteamine-SHs at the reaction center with iodoacetoamide and chloroacetyl-CoA, respectively. Dissociation and stoichiometric recombination of the unmodified and the SH-modified enzyme subunits yielded a combination of unmodified-unmodified, unmodified-modified and modified-modified hybrid dimers that together exhibit 50% activity. In contrast, hybrid dimers obtained by reconstruction of the two modified enzymes showed essentially no catalytic activity. These results suggest that the two subunits of 6-hydroxymellein synthase are aligned in head-to-tail orientation to organize two reaction centers which are comprised of a cysteine and a complementary cysteamine SH group, belonging to and contributed from the same subunit in the homodimer structure.

Journal Article↗
PubMed2000

The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase.

We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation. The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae. The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S. cerevisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system. We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening.

Amino Acid Sequence↗
PubMed2000

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis.

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.

Amino Acid Sequence↗
PubMed2000

Functional characterization of the Candida albicans MNT1 mannosyltransferase expressed heterologously in Pichia pastoris.

The alpha1,2-mannosyltransferase gene MNT1 of the human fungal pathogen Candida albicans has been shown to be important for its adherence to various human surfaces and for virulence (Buurman, E. T. , Westwater, C., Hube, B., Brown, A. P. J., Odds, F. C., and Gow, N. A. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7670-7675). The CaMnt1p is a type II membrane protein, which is part of a family of proteins that are important for both O- and N-linked mannosylation in fungi and which represent a distinct subclass of glycosyltransferase enzymes. Here we use heterologous expression of CaMNT1 in the methylotrophic yeast Pichia pastoris to characterize the properties of the CaMnt1p enzyme as an example of this family of enzymes and to identify key amino acid residues required for coordination of the metal co-factor and for the retaining nucleophilic mechanism of the transferase reaction. We show that the enzyme can use both Mn(2+) and Zn(2+) as metal ion co-factors and that the reaction catalyzed is specific for alpha-methyl mannoside and alpha1,2-mannobiose acceptors. The N-terminal cytoplasmic tail, transmembrane domains, and stem regions were shown to be dispensable for activity, whereas truncations to the C-terminal catalytic domain destroyed activity without markedly affecting transcription of the truncated gene.

Amino Acid Sequence↗