Search PubMed⌕ Search

Biomedical subjects

Y Anraku

Publications and source records attributed to Y Anraku.

At least 127 records · Page 7Linked to original sources
PubMed1990

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport.

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.

Animals↗
PubMed1990

Defective cation-coupling mutants of Escherichia coli Na+/proline symport carrier. Characterization and localization of mutations.

A major proline carrier in Escherichia coli encoded by the putP gene mediates proline/Na+ or Li+ symport. Proline carrier mutants with altered cation specificity were obtained by mutagenesis with nitrous acid in vitro of a plasmid carrying the wild-type putP gene. Two mutant strains harboring plasmid pMOP4135 and pMOP4141 could transport proline efficiently only in the presence of an increased concentration of sodium ion. Mutations of these plasmids, putP4135 and putP4141, caused reduction of affinity for Na+ of proline transport and binding, without remarkable change in the affinity for proline or in production of the carriers. Consistent with the lower affinity of the putP4141 carrier for Na+, the mutant carrier was supersensitive to N-ethylmaleimide inhibition. The pH dependence of proline binding was also changed in these mutant carriers. The lesions of putP4135 and putP4141 were located in the N-terminal part of the putP gene (ClaI-PvuII fragment) by in vitro recombination and subsequent examination of the phenotype of the transformants. DNA sequencing of these fragments revealed one base alteration of G to A at nucleotides 299 and 656 in pMOP4141 and pMOP4135, respectively, which corresponded to amino acid changes from Gly22 to glutamic acid and Cys141 to tyrosine, respectively.

Amino Acid Sequence↗
PubMed1990

Mechanism of Na+/proline symport in Escherichia coli: reappraisal of the effect of cation binding to the Na+/proline symport carrier.

The proton and sodium ion dependences of the proline binding and transport activities of the proline carrier in Escherichia coli were investigated in detail. The binding activity in cytoplasmic membrane vesicles from a carrier over-producing strain (PT21/pTMP5) was absolutely dependent on the presence of Na+, but did not necessarily require protonation of the carrier, in contrast to the model previously reported (Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7797-7801). Based on this and previous observations, we propose a modified model of the proline binding reaction of the proline carrier, in which a proton is supposed to be a regulatory factor for the binding activity. The apparent Michaelis constant of proline (Kt) of the transport activity of cytoplasmic membrane vesicles from the wild type E. coli strain driven by a respiratory substrate, ascorbate, showed dependence on a low concentration of sodium ion. The Michaelis constant of sodium ion for transport (KtNa) was estimated to be 25 microM. The proline transport activities in membrane vesicles and intact cells were modulated by H+ concentration, the inhibitory effect of protons (pKa approximately equal to 6) being similar to that observed previously (Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7802-7806). Based on these observations and the modified model of substrate binding to the proline carrier, a model of the proline/Na+ symport mechanism is proposed, in which a proton is postulated to be a regulatory factor of the transport activity.

Amino Acid Transport Systems, Neutral↗
PubMed1990

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity.

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.

Adenosine Triphosphatases↗
PubMed1990

The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function.

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

Amino Acid Sequence↗
PubMed1990

Novel antibiotics, furaquinocins A and B. Taxonomy, fermentation, isolation and physico-chemical and biological characteristics.

Two novel antibiotics, furaquinocins A and B were isolated from the culture broth of Streptomyces sp. KO-3988. These antibiotics possess cytocidal activities against HeLa S3 cells in vitro at concentrations of 3.1 micrograms/ml for A and 1.6 micrograms/ml for B. Neither substance possessed antimicrobial activities against Gram-positive and Gram-negative bacteria, fungi or yeast at a concentration of 1,000 micrograms/ml.

Anti-Bacterial Agents↗
PubMed1989

Orientation of the carboxyl terminus of the Na+/proline symport carrier in Escherichia coli.

The orientation of the carboxyl terminal region of the Escherichia coli proline carrier in the cytoplasmic membrane was studied. The beta-galactosidase moiety of the PutP-LacZ fusion protein [(1987) J. Biol. Chem. 262, 14100-14104] was exposed outside the inside-out vesicles and inside the right-side-out vesicles. A site-directed antibody raised against a synthetic peptide corresponding to the putative carboxyl terminal region of the carrier reacted preferentially with the inside-out vesicles prepared from a wild-type proline carrier overproducing strain and less with the right-side-out vesicles. These results indicate that the carboxyl terminus of the proline carrier is exposed to the cytoplasmic side of the membrane.

Amino Acid Transport Systems, Neutral↗
PubMed1989

Nucleotide sequence of AMS1, the structure gene of vacuolar alpha-mannosidase of Saccharomyces cerevisiae.

AMS1, a structure gene of the vacuolar membrane alpha-mannosidase of Saccharomyces cerevisiae, has been characterized and found to encode both constituent polypeptides of the enzyme, a 107 kDa polypeptide and a 73 kDa polypeptide. The nucleotide sequence of AMS1 demonstrates that the gene encodes 1083 amino acids with a molecular weight 124,497. Although the enzyme is considered to exist on the inner surface of the vacuolar membrane, the predicted primary amino acid sequence does not have a hydrophobic stretch suitable for a signal sequence in its N-terminal region.

Amino Acid Sequence↗
PubMed1989

Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport.

The lacYun gene, which encodes a lactose carrier showing the uncoupled phenotype of substrate transport in Escherichia coli [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414], was cloned on a plasmid vector, pBR322. The binding of a substrate, p-nitrophenyl alpha-galactoside, to the lacYun carrier in membranes from the strain harbouring the lacYun clone showed a pH-dependence different from its binding to the wild-type lactose carrier. This finding indicated that the lacYun mutation confers higher affinity for H+ on the carrier, exerting its effect on the less efficient dissociation of substrate inside cells. The result coincides with the proposal [Yamato & Rosenbusch (1983) FEBS Lett. 151, 102-104] that the proton affecting the substrate binding is the coupling proton of the proton/lactose symport reaction, which allows only the ordered mechanism of binding of substrate to an H+-carrier binary complex. From the simplest model of the symport reaction, constructed on the basis of these results, the coupling site of energy in the carrier cycle of the transport reaction can be identified at the substrate-dissociation step inside cells.

Binding Sites↗
PubMed1989

Functional molecular masses of vacuolar membrane H+-ATPase from Saccharomyces cerevisiae as studied by radiation inactivation analysis.

The functional molecular masses of the vacuolar membrane H+-ATPase in Saccharomyces cerevisiae under two kinetic conditions for ATP hydrolysis were measured by radiation inactivation. When vacuolar membrane vesicles were exposed to gamma-rays from 60Co, the activities catalyzing a single-cycle and multi-cycles of ATP hydrolysis both decreased as single-exponential functions of the radiation dosage. By applying the target theory, the functional molecular masses for single- and multi-cycle hydrolyses of ATP were determined to be approx. 0.9-1.1 X 10(5) and 4.1-5.3 X 10(5) Da, respectively. N,N'-Dicyclohexylcarbodiimide (DCCD) did not inhibit the former reaction but strongly inhibited the latter. It is suggested that the ATPase with a minimal composite of subunits a and b, in which subunit c is not necessarily involved operationally, can catalyze single-cycle hydrolysis of ATP, whereas for multi-cycle hydrolysis of ATP, the ATPase requires a properly organized oligomeric structure with subunits a-c, which may direct a positive cooperative mechanism of ATP hydrolysis and coupled H+ translocation in a DCCD-sensitive manner.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Functional expression of chicken calmodulin in yeast.

The coding region of a chicken calmodulin cDNA was fused to a galactose-inducible GAL1 promoter, and an expression system was constructed in the yeast Saccharomyces cerevisiae. Expression of calmodulin was demonstrated by purifying the heterologously expressed protein and analyzing its biochemical properties. When the expression plasmid was introduced into a calmodulin gene (cmd1)-disrupted strain of yeast, the cells grew in galactose medium, showing that chicken calmodulin could complement the lesion of yeast calmodulin functionally. Repression of chicken calmodulin in the (cmd1)-disrupted strain caused cell cycle arrest with a G2/M nucleus, as observed previously with a conditional-lethal mutant of yeast calmodulin. These results suggest that the essential function of calmodulin for cell proliferation is conserved in cells ranging from yeast to vertebrate cells.

Animals↗
PubMed1989

A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division.

The coding region of a yeast calmodulin gene was fused to a galactose-inducible GAL1 promoter, and a conditional-lethal mutant of Saccharomyces cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, in which the promoter was repressed, it ceased growing after 12-15 h. The growth arrest was associated with a decrease in intracellular calmodulin levels: after 12 h, no intracellular calmodulin protein was detectable. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. Thus, the defect was mainly in nuclear division. Bud growth was partially inhibited in these cells: 27% of the cells stopped growing with a small bud. Furthermore, calmodulin-deficient cells showed elevated rates of chromosome loss, possibly as the result of a defect in the precise segregation of chromosomes.

Amino Acid Sequence↗
PubMed1989

Structure and function of the yeast vacuolar membrane proton ATPase.

Our current work on a vacuolar membrane proton ATPase in the yeast Saccharomyces cerevisiae has revealed that it is a third type of H+-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F0F1-type ATP synthase and the plasma membrane E1E2-type H+-ATPase. The vacuolar membrane H+-ATPase is composed of three major subunits, subunit a (Mr = 67 kDa), b (57 kDa), and c (20 kDa). Subunit a is the catalytic site and subunit c functions as a channel for proton translocation in the enzyme complex. The function of subunit b has not yet been identified. The functional molecular masses of the H+-ATPase under two kinetic conditions have been determined to be 0.9-1.1 x 10(5) daltons for single-cycle hydrolysis of ATP and 4.1-5.3 x 10(5) daltons for multicycle hydrolysis of ATP, respectively. N,N'-Dicyclohexyl-carbodiimide2 does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-sensitive catalytic site. Cloning of structural genes for the three subunits of the H+-ATPase (VMA1, VMA2, and VMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.

Cloning, Molecular↗
PubMed1989

Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B.

Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E. coli K-12 strain by using a cosmid vector, pHC79. One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system. The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain. The gltS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with an apparent Mr of 35,000 in a minicell system. A gene designated gltP was also cloned. The transport activity of the gltP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li+. An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, monensin, inhibited only that of the gltS system. The Kt value for glutamate was 11 microM in the gltP system and 3.5 microM in the gltS system. L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gltS system. Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E. coli strain. These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system.

Blotting, Southern↗
PubMed1989

Isolation of isoflavonoids possessing antioxidant activity from the fermentation broth of Streptomyces sp.

Three antioxidant isoflavonoids characterized as 4',7,8-trihydroxyisoflavone (1), 3',4',7-trihydroxyisoflavone (2) and 8-chloro-3',4',5,7-tetrahydroxyisoflavone (3) were isolated from the cultured broth of Streptomyces sp. OH-1049. Among them, 3 is a novel isoflavonoid possessing a chlorine atom in the molecule. In in vitro studies, these antibiotics were found to possess antioxidant activity whereas showed almost no cytocidal activities against HeLa S3 cells.

Animals↗
PubMed1989

Structural study of isoflavonoids possessing antioxidant activity isolated from the fermentation broth of Streptomyces sp.

Structures of three antioxidant isoflavonoids isolated from the cultured broth of Streptomyces sp. OH-1049 were shown to be 4',7,8-trihydroxyisoflavone (1), 3',4',7-trihydroxyisoflavone (2) and 8-chloro-3',4',5,7-tetrahydroxyisoflavone (3), respectively. Among them, 3 is a novel isoflavonoid possessing a chlorine atom in the molecule. Compound 1 was synthesized and its antitumor activities were tested against IMC carcinoma, S180, P388 leukemia and P388/ADM leukemia in vivo. As a result, 1 showed 139% increase in life span (ILS) against S180 bearing mice whereas it showed slight or no ILS against IMC carcinoma, P388 leukemia and P388/ADM leukemia bearing mice.

Animals↗
PubMed1989

Novel cytocidal antibiotics, glucopiericidinols A1 and A2. Taxonomy, fermentation, isolation, structure elucidation and biological characteristics.

Two novel antibiotics, glucopiericidinols A1 (1) and A2 (2) were isolated from the cultured broth of Streptomyces sp. OM-5689. The structures of these two compounds were deduced employing spectroscopic analyses. These antibiotics showed potent cytocidal activities against HeLa S3 cells in vitro (MIC 1: 0.39 microgram/ml, 2: 0.10 microgram/ml) when the cells were exposed to the antibiotics for 3 days. Although 1 and 2 showed no activity at 1,000 micrograms/ml against various Gram-positive and Gram-negative bacteria, yeast or fungi, they did have inhibitory activity against Piricularia oryzae (MIC of 1: 125 micrograms/ml, of 2: 31 micrograms/ml).

Aminoglycosides↗