Biomedical subjects

M Arisawa

Publications and source records attributed to M Arisawa.

At least 73 records · Page 4Linked to original sources

PubMed1996

A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3.

We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.

Adenosine Triphosphatases↗

PubMed1996

Supra- and infratentorial multiple cavernous angiomas in an infant: report with MR evaluation of an unusual case.

A rare case of multiple cavernous angiomas with repeated hemorrhages in a 9-month-old male infant is presented, together with sequential computed tomography (CT) and magnetic resonance (MR) images. The infant underwent hematoma evacuation for left cerebellar hemorrhage and his postoperative course was uneventful. The multiple cavernous angiomas receded gradually without any treatment except the hematoma evacuation. Two years following presentation, he continues to do well. To the authors' knowledge, this is the first report of multiple cavernous angiomas with brain stem involvement in an infant.

Brain Neoplasms↗

PubMed1996

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis.

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.

Antibodies, Fungal↗

PubMed1996

Isolation of the mRNA-capping enzyme and ferric-reductase-related genes from Candida albicans.

The mRNA-capping enzyme (mRNA 5'-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1 delta null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1 delta null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5'-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1 delta null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.

Amino Acid Sequence↗

PubMed1996

Antifungal azoxybacilin exhibits activity by inhibiting gene expression of sulfite reductase.

Azoxybacilin, produced by Bacillus cereus, has a broad spectrum of antifungal activity in methionine-free medium and has been suggested to inhibit sulfite fixation. We have further investigated the mode of action by which azoxybacilin kills fungi. The compound inhibited the incorporation of [35S] sulfate into acid-insoluble fractions of Saccharomyces cerevisiae under conditions in which virtually no inhibition was observed for DNA, RNA, or protein synthesis. It did not interfere with the activity of the enzymes for sulfate assimilation but clearly inhibited the induction of those enzymes when S. cerevisiae cells were transferred from rich medium to a synthetic methionine-free medium. Particularly strong inhibition was observed in the induction of sulfite reductase. Northern (RNA) analysis revealed that azoxybacilin decreased the level of mRNA of genes for sulfate assimilation, including MET10 for sulfite reductase and MET4, the transactivator of MET10 and other sulfate assimilation genes. When activities of azoxybacilin were compared for mRNA and enzyme syntheses from MET10, the concentration required for inhibition of transcription of the gene was about 10 times higher (50% inhibitory concentration = 30 micrograms/ml) than that required for inhibition of induction of enzyme synthesis (50% inhibitory concentration = 3 micrograms/ml). The data suggest that azoxybacilin acts on at least two steps in the expression of sulfite reductase; the transcriptional activation of MET4 and a posttranscriptional regulation in MET10 expression. We conclude that azoxybacilin exhibits antifungal activity by interfering with the regulation of expression of sulfite reductase activity.

Aminobutyrates↗

PubMed1996

HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae.

Bacterial Outer Membrane Proteins↗

PubMed1996

Role of three chitin synthase genes in the growth of Candida albicans.

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants.

Animals↗

PubMed1996

ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.

Base Sequence↗

PubMed1996

Signaling toward yeast 1,3-beta-glucan synthesis.

1,3-beta-glucan synthase catalyzes the synthesis of a 1,3-beta-linked glucan polymer which produces the main rigidity of the yeast cell wall. Recent success in purification of this enzyme by product entrapment (21) has provided new insights into the dynamic aspects of the cell wall. This relatively simple procedure made it possible to identify the genes encoding the catalytic subunits of glucan synthase. In addition, the involvement of a rho type GTPase in the regulation of glucan synthase was demonstrated with the purified enzyme. Based on intracellular localization of the glucan synthase subunits, we have proposed a model in which assembly of the subunits is important for the activation of glucan synthase at sites of polarized growth. In this article, we will focus on biochemistry of 1,3-beta-glucan synthase and signaling through rho type GTPase.

Fungal Proteins↗

PubMed1996

[Assessment of degree of edema by means of bioelectrical impedance during pregnancy].

It is well known that the total body water in women with toxemia of pregnancy expands beyond the normally increased volume that characterizes pregnancy. Although abnormally increased water retention is usually diagnosed by both the edema of the legs and the weight gain, these methods are not quantitative. To quantify the degree of edema, we used bioelectrical impedance analysis (BIA). Because water and electrolytes are the dominant factors affecting electrical conduction in the body, the increase in total body water (the degree of edema) is assessed by BIA. BIA was conducted serially in a group of 38 normal pregnant women during pregnancy and 4 pregnant women with toxemia of pregnancy. The values for bioelectrical impedance in normal pregnant women decreased gradually during pregnancy, suggesting a physiological increase in total body water (p < 0.01). In contrast, the values for bioelectrical impedance dramatically decreased in the cases of 4 women with toxemia of pregnancy and accurately increased in the process of recovery from toxemia of pregnancy. These findings indicate that bioelectrical impedance analysis is a useful and practical method for assessing the increase in total body water.

Adult↗

PubMed1995

Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation.

The Candida glabrata (Cg) TRP1 and HIS3 genes have been isolated by complementation of the Saccharomyces cerevisiae (Sc) trp1 and his3 mutants, respectively. Cg TRP1 encodes a polypeptide of 217 amino acids (aa), whose aa sequence is 58% identical to that of Sc TRP1. Cg HIS3 encodes a polypeptide of 210 aa, whose aa sequence is 73% identical to that of the Sc HIS3. Both Cg TRP1 and HIS3 were disrupted by sequential integrative transformation where the Sc URA3 was used as a selection marker for transformation. The resulting auxotrophic strain of his3- and trp1- was used to examine the ability of the Sc genes to complement the Cg mutations; Sc HIS3 and TRP1 complemented the Cg his3- and trp1- mutations, respectively.

Aldose-Ketose Isomerases↗

PubMed1995

Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae.

1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.

Amino Acid Sequence↗

PubMed1995

Characterization of the binding site for cyclothialidine on the B subunit of DNA gyrase.

The mechanism of inhibition of DNA gyrase by cyclothialidine, a novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using [14C]benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit, the B subunit and relaxed ColE1 DNA. The mechanism of inhibition was also studied with the 43-kDa N-terminal fragment of the B subunit. The [14C]benzoylcyclothialidine could bind to the B subunit alone but not to the A subunit nor to the plasmid DNA alone. Furthermore, the compound also bound to the 43-kDa N-terminal fragment of the B subunit. Scatchard analysis of [14C]benzoylcyclothialidine binding to DNA gyrase showed that the binding affinity of the compound increased, depending on the assembly of the gyrase (A2B2). DNA complex. This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyrase.DNA complex (increase in affinity: B-->A2B2-->A2B2.DNA). Furthermore, displacement curves of [14C]benzoylcyclothialidine binding by nonlabeled cyclothialidine, ATP analogues, and coumarin antibiotics indicated that cyclothialidine, coumarins, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ.

Adenosine Triphosphatases↗

PubMed1995

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites.

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.

Amino Acid Sequence↗

PubMed1995

Cloning and sequencing of sulfite reductase alpha subunit gene from Saccharomyces cerevisiae.

A DNA fragment of 2.1 kb was specifically amplified by PCR with primers based on the amino acid sequences obtained from the N-terminal region and the cyanogen bromide-derived peptide of the sulfite reductase alpha subunit in Saccharomyces cerevisiae. With this fragment as a probe, the gene coding for the sulfite reductase alpha subunit was isolated from a genomic library of S. cerevisiae. Sequencing analysis revealed that the gene contains a 3105-bp open reading frame, which is large enough to code for a protein of 1035 amino acid residues. The transcript of the sulfite reductase alpha subunit gene was detected by Northern analysis after methionine deprivation, but the amount of the transcript did not directly correlate with the enzyme activity. The DNA fragment containing the sulfite reductase alpha subunit gene rescued the met10 phenotype by complementation.

Amino Acid Sequence↗

PubMed1995

Isolation of canCHS1A, a variant gene of Candida albicans chitin synthase.

A canCHS1A gene encoding the chitin synthase of Candida albicans was cloned. DNA sequencing and comparison with another canCHS1 gene described elsewhere indicated that the canCHS1A gene encoded a polypeptide with 775 amino acid residues, a protein with one less amino acid than that encoded by the canCHS1 gene. A six-base alteration was observed between the two genes, suggesting that the canCHS1A gene is a variant gene of the canCHS1. The pH profile/activity relationship of canChs1A in permeabilized cells was identical to that of canChs1. The canChs1A enzyme was competitively inhibited by polyoxin D (5.2 microM) and nikkomycin Z (12 microM). When the cloned gene was expressed in a Saccharomyces cerevisiae chs2 mutant that exhibited aberrant morphology, the normal structure was restored. We conclude that the function of the canCHS1A gene is similar to that of sacCHS2 in S. cerevisiae.

Aminoglycosides↗

PubMed1995

Random amplified polymorphic DNA analysis of clinically and environmentally isolated Cryptococcus neoformans in Nagasaki.

We examined clinical (pulmonary cryptococcosis and cryptococcal meningitis) and environmental (pigeon excreta) isolates of Cryptococcus neoformans var. neoformans (serotype A) in the southern Japanese prefecture of Nagasaki. The random amplified polymorphic DNA profiles obtained by using three primers revealed six patterns among 21 clinical isolates and three patterns among 8 environmental isolates. Pattern I was the most common (18 of 29 isolates) and was found among isolates obtained throughout the entire Nagasaki Prefecture. Patterns I, III, and IV were found among both clinical and environmental isolates. Patterns I and IV had a characteristic distribution, and in particular, pattern IV was isolated exclusively (five of six isolates) from isolates from Nagasaki City. Two environmental isolates from two locations associated strongly with two patients revealed identical random amplified polymorphic DNA patterns (patterns I and IV) for isolates from each patient. Our results suggest that clinical and environmental isolates belong to the same pool of C. neoformans isolates and that these isolates have certain geographic locations, although the number of isolated strains was limited.

Adolescent↗

PubMed1995

Design of an antifungal methionine inhibitor not antagonized by methionine.

Only a few biosynthetic pathways in fungal cells have been used as antifungal targets. Therefore, the number of antifungals has been limited, and a cross-drug resistance among them has emerged in the therapy of mycoses. Under such circumstances, the identification of an antifungal with a new mode of action is highly desirable. By infecting mice with a mutant of C. albicans deficient in the sulfate assimilation pathway, we have discovered a new target for the discovery of antifungal agents. We have proven that azoxybacilin inhibits the sulfate assimilation pathway by showing its inhibitory activity for [35S]SO4 incorporation into proteins. We have also demonstrated that azoxybacilin was taken up into fungal cells via an active transport system specific for methionine. This sharing of the uptake system with methionine may explain the mechanism by which the antifungal activity of azoxybacilin is antagonized by methionine, and led us to design azoxybacilin derivatives that lack the structural feature of amino acids and, at the same time, have increased hydrophobicity to give higher non-specific permeability through the cell membrane. As a result, we have found that ester derivatives of azoxybacilin were not antagonized by methionine in their uptake, and that they showed antifungal activity independent of methionine. The benzyl ester of azoxybacilin was the same as azoxybacilin in its mode of action, but was not markedly antagonized by methionine at concentrations up to 1 mg/ml. These results suggest that azoxybacilin may not merely interfere with the sulfate assimilation pathway.

Aminobutyrates↗