Nitric oxide form of a pyridylphenylurea is a potent inducer of shoot formation in plant tissue cultures.
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Cell Differentiation↗
Biomedical subjects
M Arisawa
Publications and source records attributed to M Arisawa.
Homonojirimycin analogues and their glucosides from Lobelia sessilifolia and Adenophora spp. (Campanulaceae).
2,6-Dideoxy-7-O-(beta-D-glucopyranosyl) 2,6-imino-D-glycero-L-gulo- heptitol (7-O-beta-D-glucopyranosyl-alpha-homonojirimycin, 1) was isolated from the 50% methanol extract of the whole plant of Lobelia sessilifolia (Campanulaceae), which was found to potently inhibit rice alpha-glucosidase. Adenophorae radix, roots of Adenophora spp. (Campanulaceae), yielded new homonojirimycin derivatives, adenophorine (2), 1-deoxyadenophorine (3), 5-deoxyadenophorine (4), 1-C-(5-amino-5-deoxy-beta-D-galactopyranosyl)butane (beta-1-C-butyl-deoxygalactonojirimycin, 5), and the 1-O-beta-D-glucosides of 2 (6) and 4 (7), in addition to the recently discovered alpha-1-C-ethylfagomine (8) and the known 1-deoxymannojirimycin (9) and 2R,5R-bis(hydroxymethyl)-3R,4R- dihydroxypyrrolidine (DMDP, 10). Compound 4 is a potent inhibitor of coffee bean alpha-galactosidase (IC50 = 6.4 microM) and a reasonably good inhibitor of bovine liver beta-galactosidase (IC50 = 34 microM). Compound 5 is a very specific and potent inhibitor of coffee bean alpha-galactosidase (IC50 = 0.71 microM). The glucosides 1 and 7 were potent inhibitors of various alpha-glucosidases, with IC50 values ranging from 1 to 0.1 microM. Furthermore, 1 potently inhibited porcine kidney trehalase (IC50 = 0.013 microM) but failed to inhibit alpha-galactosidase, whereas 7 was a potent inhibitor of alpha-galactosidase (IC50 = 1.7 microM) without trehalase inhibitory activity.
1-Deoxynojirimycin↗
Up-regulation of genes encoding glycosylphosphatidylinositol (GPI)-attached proteins in response to cell wall damage caused by disruption of FKS1 in Saccharomyces cerevisiae.
FKS1 and FKS2 encode alternative catalytic subunits of the glucan synthases that are responsible for synthesis of beta-1,3-glucan in the Saccharomyces cerevisiae cell wall. Disruption of FKS1 reduces the glucan content of the cell wall, increases chitin content and activates the expression of CWP1, which encodes a glycosylphosphatidylinositol (GPI)-dependent cell wall protein. These cellular responses have been regarded as compensating for cell wall damage in order to maintain cell wall integrity. Here, we report the identification, by genome-wide screening, of 22 genes that are transcriptionally up-regulated in fks1delta cells. Among them, five genes were found to encode GPI-attached proteins, three of which are covalently associated with the cell wall. Deletion and replacement analysis of the promoter regions identified Rlm1-binding sequences as being responsible for the up-regulation following disruption of FKS1. Using the rlm1delta tetOp-FKS1 strain, in which the expression of FKS1 can be repressed by doxycycline, we examined the requirement for Rlm1 for the transcriptional up-regulation of these five genes. Three of the five genes were not up-regulated by doxycycline, indicating that Rlm1 mediates their up-regulation when FKS1 is inactivated. The remaining two genes were up-regulated by doxycycline, suggesting that a transcription factor other than Rlm1 is involved in their response to disruption of FKS1.
Aspartic Acid Endopeptidases↗
Hypotensive action of Nangapiry, a Paraguayan natural medicine, in rodents.
Hypotensive action mechanism of a cation exchange resin adsorbate (IR-120A) separated from a Paraguayan Natural Medicine, Nangapiry, was investigated. Blood pressures of normal and pithed rats and contractions of isolated thoracic aorta and atria of mice were measured. The blood pressure on normal rats was reduced by an intravenous injection of IR-120A (5 mg/kg). The hypotensive effect on the pithed rat appeared more lasting than that on normal rats by IR-120A. The IR-120A (100 microg-3 mg/ml) concentration-dependently depressed prostaglandin (PG) F2alpha (10 microM)- or KCl (40 mM)-induced aortic contractions and electrically-evoked contraction of left atria, and at a lesser extent spontaneous beating rate of right atria. The 50% inhibitory concentrations (IC50) for the PGF2alpha- and KCl-induced aortic contractions were 713 and 828 microg/ml, respectively, and the IC50 values for the muscle contraction and the beating rate were 1.04 and >3 mg/ml, respectively. These results suggest that the hypotensive action of IR-120A are peripherally elicited by the dilatation of artery and the depression of heart contraction, but not the reduction of heart rate.
Animals↗
Depletion of the squalene synthase (ERG9) gene does not impair growth of Candida glabrata in mice.
Squalene synthase (farnesyl-diphosphate farnesyltransferase, EC 2.5. 1.21) is the first committed enzyme of the sterol biosynthesis pathway. Inhibitors of this enzyme have been intensively studied as potential antifungal agents. To assess the effect of deactivating squalene synthase on the growth of fungi in mice, we isolated the squalene synthase (ERG9) gene from the pathogenic fungus Candida glabrata and generated strains in which the CgERG9 gene was under the control of the tetracycline-regulatable promoter. Depletion of the ERG9 gene by doxycycline (DOX), a derivative of tetracycline, decreased the cell viability in laboratory media, whereas it did not affect cell growth in mice at all. The growth defect caused by DOX in laboratory media was suppressed by the addition of serum. Analyses of the sterol composition of the restored cells in serum-containing media suggest that the defect of ergosterol biosynthesis can be complemented by the incorporation of exogenous cholesterol into the cells. Thus, deactivation of squalene synthase did not affect fungal growth in mice, presumably because the cells were able to incorporate cholesterol from the serum. These results showed that squalene synthase could not be a suitable target of antifungals for the treatment of C. glabrata infection.
Amino Acid Sequence↗
Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans.
Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.
Animals↗
Construction of chiral 1,2-cycloalkanopyrrolidines from L-proline using ring closing metathesis (RCM).
An efficient synthetic method for the preparation of optically active pyrroloazocine, pyrroloazepine, quinolizidine, indolizidine using ring closing olefin metathesis (RCM) is described.
Molecular Structure↗
[Development of intramolecular oxidative phenolic coupling reactions using hypervalent iodine (III) reagents and their application to the synthesis of Amaryllidaceae alkaloids].
Hypervalent iodine (III) reagents nowadays are used extensively in the field of organic chemistry. Especially, phenyliodine (III) diacetate (PIDA) or phenyliodine (III) bis(trifluoroacetate) (PIFA) have received much attention because of their reactivities similar to heavy metal reagents or anodic oxidation, low toxicity, ready availability and easy handling. In the continuous study of our oxidative phenolic coupling reactions using a hypervalent iodine (III) reagents, a versatile synthetic procedure for the galanthamine-type Amaryllidaceae alkaloids was accomplished. The first total synthesis of (+/-)-sanguinine and the total syntheses of (+/-)-galanthamine, (+/-)-narwedine, (+/-)-lycoramine, and (+/-)-norgalanthamine were also successfully carried out.
Alkaloids↗
Prenylation of Rho1p is required for activation of yeast 1, 3-beta-glucan synthase.
One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeast Saccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1, 3-beta-glucan synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the beta subunit of geranylgeranyl transferase type I called cal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-beta-glucan synthase. The 1,3-beta-glucan synthase activity of the cal1-1 membrane was significantly reduced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1, 3-beta-glucan synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. Glutathione S-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction. The modified form of GST-Rho1p was specifically able to restore the 1,3-beta-glucan synthase of the rho1-3 membrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p. These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-beta-glucan synthase in vitro. Increased cytoplasmic levels of divalent cations such as Ca(2+) restored both Rho1p modification and the 1,3-beta-glucan synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.
Base Sequence↗
Improving effects of the extracts from Eugenia uniflora on hyperglycemia and hypertriglyceridemia in mice.
EtOH (70%) extracts from the leaves of Eugenia uniflora were separated into six fractions with different polarity and molecular size, i.e. NP-1-NP-6. In an oral glucose tolerance test, NP-1 and 4 inhibited the increase in plasma glucose level. However, in an intraperitoneal glucose tolerance test, such an inhibitory effect was not seen. Thus, the effects of NP-1 and 4 were apparently due to the inhibition of glucose absorption from the intestine. In a sucrose tolerance test, all fractions inhibited the increase in plasma glucose level. In an oral corn oil tolerance test, NP-3 and 4 showed an inhibitory effect on the increase in plasma triglycerides level. On the other hand, NP-3, 4, 5 and 6 inhibited maltase and sucrase activities and all fractions except for NP-1 showed an inhibitory effect on lipase activity dose-dependently. The inhibition of the increase in plasma glucose level by NP-3, 4, 5 and 6 in the oral sucrose tolerance test and the inhibition of the increase in plasma triglycerides by NP-3 and 4 in the oral corn oil tolerance test were apparently due to the inhibition of the decomposition of carbohydrates and fats in the intestine, respectively.
Animals↗
Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis.
The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased Km value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.
Acetyltransferases↗
Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis.
Helicase-related proteins play important roles in various cellular processes incuding DNA replication, DNA repair, RNA processing and so on. It has been well known that the amino acid sequences of these proteins contain several conserved motifs, and that the open reading frames (ORFs) which encode helicase-related proteins make up several gene families. In this study, we have identified 134 ORFs that encode helicase-like proteins in the Saccharomyces genome, based on similarity with the ORFs of authentic helicase and helicase-related proteins. Multiple alignment of the ORF sequences resulted in the 134 ORFs being classified to 11 clusters. Seven out of 21 previously uncharacterized ORFs (YDL031w, YDL070w, YDL084w, YGL150c, YKL078w, YLR276c, and YMR128w) were identified by systematic gene disruption, to be essential for vegetative growth. Three (YDR332w, YGL064c, and YOL095c) out of the remaining 14 dispensable ORFs exhibited the slow-growth phenotype at 30 degrees C and 37 degrees C. Furthermore, the expression profiles of transcripts from 43 ORFs were examined under seven different growth conditions by Northern analysis and reverse transcription-polymerase chain reaction, indicating that all of the 43 tested ORFs were transcribed. Interestingly, we found that the level of transcript from 34 helicase-like genes was markedly increased by heat shock. This suggests that helicase-like genes may be involved in the biosynthesis of nucleic acids and proteins, and that the genes can be transcriptionally activated by heat shock to compensate for the repressed synthesis of mRNA and protein.
Amino Acid Sequence↗
Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel.
CLC-K1 is a kidney-specific chloride channel that mediates transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) in the inner medulla. Transport of NaCl in the tAL is thought to be a component of urinary concentration in a passive model of the countercurrent multiplication system, but there has been no direct evidence that CLC-K1 is involved in urine concentration. To analyse the physiological function of CLC-K1 in vivo, we generated mice lacking CLC-K1 by targeted gene disruption. Clcnk1-/- mice were physically normal appearance, but produced approximately five times more urine than Clcnk1+/- and Clcnk1+/+ mice. After 24 hours of water deprivation, Clcnk1-/- mice were severely dehydrated and lethargic, with a decrease of approximately 27% in body weight. Intraperitoneal injection of the V2 agonist 1-deamino-8-D-arginine vasopressin (dDAVP) induced a threefold increase in urine osmolarity in Clcnk1+/- and Clcnk1+/+ mice, whereas only a minimal increase was seen in Clcnk1-/- mice, indicating nephrogenic diabetes insipidus. After in vitro perfusion of the tAL, the lumen-to-bath chloride gradient did not produce a diffusion potential in Clcnk1-/- mice in contrast to Clcnk1+/+ and Clcnk1+/- mice. These results establish that CLC-K1 has a role in urine concentration, and that the countercurrent system in the inner medulla is involved in the generation and maintenance of hypertonic medullary interstitium.
Animals↗
Amino acid residues in the omega-minus region participate in cellular localization of yeast glycosylphosphatidylinositol-attached proteins.
The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the omega site) and Y or N at the site 2 amino acids upstream of the omega site for cell wall localization and (ii) dibasic residues in the region upstream of the omega site (the omega-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the omega-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.
Amino Acid Sequence↗
Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans.
The pathogenic fungus Candida albicans harbors three histidine kinase genes called CaSLN1, CaNIK1, and CaHK1. The disruption of any one of these three genes impaired the hyphal formation and attenuated the virulence of C. albicans in a mouse systemic candidiasis model. The effects of the disruption on hyphal formation and virulence were most severe in the cahk1Delta null mutants. Although the double disruption of CaSLN1 and CaNIK1 was impossible, further deletion of CaSLN1 or CaNIK1 in the cahk1Delta null mutants partially restored the serum-induced hypha-forming ability and virulence. When incubated with radiolabelled ATP, the recombinant CaSln1 and CaNik1 proteins, which contained their own kinase and response regulator domains, were autophosphorylated, whereas CaHk1p was not. These results imply that in C. albicans, CaSLN1 and CaNIK1 function upstream of CaHK1 but are in distinct signal transmission pathways.
Animals↗
Epidemiology of visceral mycoses: analysis of data in annual of the pathological autopsy cases in Japan.
The data on visceral mycoses that had been reported in the Annual of the Pathological Autopsy Cases in Japan from 1969 to 1994 by the Japanese Society of Pathology were analyzed epidemiologically. The frequency of visceral mycoses among the annual total number of pathological autopsy cases increased noticeably from 1.60% in 1969 to a peak of 4.66% in 1990. Among them, the incidences of candidiasis and aspergillosis increased the most. After 1990, however, the frequency of visceral mycoses decreased gradually. Until 1989, the predominant causative agent was Candida, followed in order by Aspergillus and Cryptococcus. Although the rate of candidiasis decreased by degrees from 1990, the rate of aspergillosis increased up to and then surpassed that of candidiasis in 1991. Leukemia was the major disease underlying the visceral mycoses, followed by solid cancers and other blood and hematopoietic system diseases. Severe mycotic infection has increased over the reported 25-year period, from 6.6% of the total visceral mycosis cases in 1969 to 71% in 1994. The reasons for this decrease of candidiasis combined with an increase of aspergillosis or of severe mycotic infection might be that (i) nonsevere (not disseminated) infections were excluded from the case totals, since they have become controllable by antifungal drugs such as fluconazole, but (ii) the available antifungal drugs were not efficacious against severe infections such as pulmonary aspergillosis, and (iii) the number of patients living longer in an immunocompromised state had increased because of developments in chemotherapy and progress in medical care.
AIDS-Related Opportunistic Infections↗
Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae. Identification of additional amino acid residues involved in its catalytic activity.
Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p. Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme. Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S. cerevisiae Chs1p and Chs2p. Alanine substitutions for the conserved amino acids in con2 identified five amino acids, Asn797, His799, Asp800, Trp803, and Thr805, the mutation of which severely diminished enzymatic activity and the enzyme's ability to rescue the yeast chs2 delta chs3 delta null mutant strain. Although the activities of some of the mutant enzymes were too low to measure enzyme kinetics, most of the alanine mutations in con2 affected the kcat values rather than the K(m) values. Whereas a conservative mutation of Asn797 restored the activity, those of His799, Asp800, Trp803, and Thr805 did not. A fine alignment of the amino acid sequences of con2 and Chs3p revealed that Asp800, Trp803 and Thr805 are completely conserved near the C-terminal ends of Chs3p and its homologs in other fungi. On the basis of these findings, we propose that Asp800, Trp803, and Thr805 in con2 are additional residues involved in catalysis, and hypothesise that Asp800 together with the previously identified Asp441 and Asp562 serve as polar residues necessary for the acid-based catalytic reaction of chitin synthase.
Amino Acid Sequence↗
Amino acid sequence requirement for efficient incorporation of glycosylphosphatidylinositol-associated proteins into the cell wall of Saccharomyces cerevisiae.
During cell wall biogenesis in Saccharomyces cerevisiae, some glycosylphosphatidylinositol (GPI)-attached proteins are detached from GPI moieties and bound to beta-1,6-glucan of the cell wall. The amino acid sequence requirement for the incorporation of GPI-attached proteins into the cell wall was studied by using reporter fusion proteins. Only the short omega-minus region composed of five amino acids, which is located upstream of the omega site for GPI attachment, determined the cellular localization of the GPI-associated proteins. Within the omega-minus region, amino acid residues at the omega-4 or -5 and omega-2 sites were important for the cell wall incorporation. Yap3p, a well characterized GPI-anchored plasma membrane aspartic protease, was localized in the cell wall when the omega-minus region was mutated to sequences containing Val or Ile at the omega-4 or -5 site and Val or Tyr at the omega-2 site.
Amino Acid Sequence↗