Search PubMed⌕ Search

Biomedical subjects

S Ohnuma

Publications and source records attributed to S Ohnuma.

At least 37 records · Page 2Linked to original sources
PubMed1998

A pathway where polyprenyl diphosphate elongates in prenyltransferase. Insight into a common mechanism of chain length determination of prenyltransferases.

Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase. The side chains of these amino acids are situated on the same side of an alpha-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996) J. Biol. Chem. 271, 18831-18837). A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40. A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40 compound with C35 and C45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.

Alkyl and Aryl Transferases↗
PubMed1998

Recognition of allylic substrates in Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase: analysis using mutated enzymes and artificial allylic substrates.

We examined the substrate specificity of two mutated geranylgeranyl diphosphate synthases, I-9 and I-11, with respect to several artificial substrates. These mutated enzymes have replacements in the amino acid sequences from positions 170 to 173, which are thought to be a part of the putative substrate binding region. The wild-type enzyme catalyzes the condensation of IPP with a series of (2E)-3-methyl-2-alkenyl diphosphates to give products with carbon numbers between 14 and 21. On the other hand, the mutated enzymes show lower activities for artificial substrates with short alkyl chains than those of the wild-type enzyme though the carbon numbers of the products are similar to those in the case of the wild-type. The mutated enzyme I-11 never accepts artificial substrates shorter than C8. Analysis of additional mutated enzymes revealed that the characteristics of the mutated enzymes arise from a few substitutions within positions 171 to 173. These results indicate that the amino acids in the positions 171 to173 of the geranylgeranyl diphosphate synthase from Sulfolobus acidocaldarius are involved in recognition of short allylic substrates, such as dimethylallyl diphosphate, but not in recognition of the chain length of the products.

Alkyl and Aryl Transferases↗
PubMed1998

Identification of genes affecting lycopene formation in Escherichia coli transformed with carotenoid biosynthetic genes: candidates for early genes in isoprenoid biosynthesis.

Although isopentenyl diphosphate is a precursor of isoprenoids in Escherichia coli, the genes and enzymes involved in its biosynthesis have not been identified. Thus, we tried to isolate E. coli mutants deficient in the biosynthesis and their complementary genes by use of an artificial phenotypic screening system employing three carotenoid biosynthetic genes, crtE, crtB, and crtI. Cells were mutagenized with ethylmethanesulfonate, then transformed with a plasmid for expression of the carotenogenic genes. Mutants deficient in biosynthesis of isopentenyl diphosphate were expected to form white colonies, because they are unable to produce enough lycopene, whereas wild-type cells form red colonies. Among large numbers of red colonies, we identified 117 white colonies. Next, we transformed each mutant with an E. coli genomic library. Twenty-nine complementary genes that restore red color of host colonies were isolated. A homology search and further complementation study using subcloned genes revealed that the true complementary genes encode isopentenyl diphosphate isomerase, subunits of ATP synthase, enzymes of the Krebs cycle, some aldehyde dehydrogenases, phosphate acetyltransferase, and enzymes which relate to the biosynthesis of ubiquinones and menaquinones. Two unknown genes were also found, designated elb1 and 2, which may be involved in the early steps of isoprenoid biosynthesis.

Carotenoids↗
PubMed1998

Overexpression of an archaeal geranylgeranyl diphosphate synthase in Escherichia coli cells.

An archaeal geranylgeranyl diphosphate synthase was overexpressed in Escherichia coli cells as fusion proteins. These fusion proteins retained their thermostability and had higher specific activity than did a partially purified native enzyme Previously reported. We purified 24.3 mg of MBP (maltose-binding protein)-fusion protein and 5.4 mg of GST (glutathione S-transferase)-fusion protein from a one-liter culture of E. coli. The MBP-fusion proteins existed in dimer, tetramer, octamer, or dodecamer form, and their product specificities were altered according to the oligomerization. The MBP-fusion protein has protease-sensitive sites in the portion corresponding to geranylgeranyl diphosphate synthase.

ATP-Binding Cassette Transporters↗
PubMed1997

High sialic acid reactivity of sugar chain structure Lewis-X in patients with mental retardation.

It has been reported that platelet-derived growth factor B-chains homodimer (PDGF-BB) improves learning function of mice, and that sugar chain structure Lewis-X of N-glycosylated glycoprotein promotes PDGF-BB secretion from platelets. Based on these findings, we assumed that learning dysfunction in some patients with mental retardation might be due to abnormality in PDGF-BB metabolism and/or Lewis-X structure. No difference in the reactivity of PDGF-BB and Lewis-X was found between the serum of patients with mental retardation and that of normals. But sialic acid reactivity of the Lewis-X fraction in some patients was remarkably higher than that in other patients and in normals. These findings suggest that sialic acids in the Lewis-X fraction may have a relation to one of the causes of learning dysfunction in these patients.

Adult↗
PubMed1997

Effects of random mutagenesis in a putative substrate-binding domain of geranylgeranyl diphosphate synthase upon intermediate formation and substrate specificity.

Archaeal geranylgeranyl diphosphate (GGPP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce GGPP with significant amounts of intermediates. To obtain information about the amino acids involved in the condensation and the release of intermediates, we randomly mutagenized two proximal regions, I and II, of the Sulfolobus acidocaldarius GGPP synthase gene and created two degenerate libraries, I and II, respectively. Regions I and II correspond to amino acid residues 170-173 and 166-168, respectively. The prenyltransferase activities of about 200 clones were analyzed using the in vivo red-white system and the conventional in vitro assay. Although, in library I, no mutated enzymes that failed to catalyze the formation of GGPP were found, as assayed with the red-white system, almost all the mutated enzymes exhibited weak GGPP synthesis activity, and many produced large amounts of intermediates. The formation of intermediates increased as the concentration of IPP was decreased or as the concentration of the allylic substrate was increased. These phenomena can be regarded as a reflection of the increased K(m) for IPP and the decreased affinity for products including intermediates. On the other hand, no mutants from library II showed such changes. These results suggest that the region from 170 to 173 is concerned in the recognition of both IPP and allylic diphosphates, and that the change in responsiveness to prenyl diphosphates causes a change in intermediate formation.

Alkyl and Aryl Transferases↗
PubMed1997

[The roles of various media in the decision making processes for recycling behavior: a path analysis model].

This study researched the effects of cognitive variables on recycling behavior, as well as effects of various media of influence on the cognition and behavior. According to Hirose (1994), the decision making process for recycling consists of two steps. The first leads to goal intention of an ecological lifestyle. The second is related to behavior intention of recycling in line with the goal intention. Mass media, such as newspaper and TV, are thought to influence beliefs about environmental problems, including three determinants of goal intention: perception of seriousness, responsibility, and effectiveness. Personal media, such as personal contacts with pro-environmental activists, are thought to influence evaluation of behavior, including three determinants of behavior intention: evaluation of feasibility, cost and benefits, and social norms. Local media, such as municipal announcement and circular, are hypothesized to have a mixed effect of the two. Path analysis indicated that goal intention affected recycling behavior through behavior intention. Effects of the three media of influence on the cognitive variables were also consistent with the hypothesis.

Behavior↗
PubMed1996

Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification.

A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303. The gene mapped at 10.9 min on the E. coli chromosome and was designated fsr (fosmidomycin resistance). Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa. A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol. Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments. The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr. The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid. These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E. coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.

Amino Acid Sequence↗
PubMed1996

Conversion of product specificity of archaebacterial geranylgeranyl-diphosphate synthase. Identification of essential amino acid residues for chain length determination of prenyltransferase reaction.

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH3, which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from approximately 1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T.(1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.

Alcohols↗
PubMed1996

Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis.

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.

Alkyl and Aryl Transferases↗
PubMed1996

Involvement of cell-mediated killing in apoptosis in histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto disease).

Histiocytic necrotizing lymphadenitis, also called Kikuchi-Fujimoto (KF) disease, is a benign disorder characterized histologically by paracortical necrotic foci surrounded by histiocytic aggregates. We analysed affected lymph node tissues from 34 patients with the disease in an attempt to elucidate its histogenesis. The 'necrotizing' cells showed typical apoptotic changes, including cell shrinkage and condensed and fragmented nuclei. Apoptotic bodies with a peculiar ultrastructure were demonstrated, and DNA fragmentation was detected in these cells by in situ end labelling. Immunostaining for the apoptosis-regulating proteins bcl-2, bax, c-myc and p53 failed to show their involvement in KF disease. However, perforin, a killer cell-specific cytolytic protein essential for provoking apoptosis in target cells, was found to be expressed abundantly by the infiltrating cells, which were thought to be cytotoxic T-lymphocytes. Perforin-expressing cells were present in the apoptotic foci of 28 of the 34 patients (82.4%). Virtually no cells containing perforin granules were present in non-pathological regions, lymph node tissues from control subjects with reactive or tuberculous lymphadenitis or those from patients with KF disease with negligible apoptosis. Therefore, the 'necrosis' associated with KF disease appears to be attributable to trans apoptotic death of the killer cell target in the affected nodes. We propose that KF disease should be called apoptotic lymphadenitis.

Adolescent↗
PubMed1996

Identification and characterization of geranylgeraniol kinase and geranylgeranyl phosphate kinase from the Archaebacterium Sulfolobus acidocaldarius.

Geranylgeranyl diphosphate is an important precursor of archaebacterial ether-linked lipids, and it has been thought that all of this compound is "de novo" synthesized by geranylgeranyl diphosphate synthase. We studied the phosphorylation of geranylgeraniol, which seems to be related to the salvage pathway of biosynthesis of archaebacterial ether-linked lipids, in the Archaebacterium Sulfolobus acidocaldarius. Activities of geranylgeraniol kinase and geranylgeranyl phosphate kinase were detected in a cell lysate of S. acidocaldarius. The two enzymes were easily separated by ultracentrifugation. The membrane fraction and the cytosolic fraction contained geranylgeraniol kinase activity and geranylgeranyl phosphate kinase activity, respectively. Geranylgeraniol kinase, which requires divalent cation such as Mg2+, Co2+, and Mn2+ and NTP (ATP, GTP, CTP, UTP), catalyzes monophosphorylation of (all-E)-geranylgeraniol to produce geranylgeranyl phosphate. (all-E)-Farnesol, (all-E)-hexaprenol, and (all-E)-octaprenol were also active substrates, though they were less effective than (all-E)-geranylgeraniol. However, neither geraniol nor (22E,6E,10Z,14Z,18Z,22Z,26Z,++ +30Z,34Z,38Z)-undecaprenol was active. This enzyme is extremely thermostable and its pH optimal is between 6.5 and 8.5. The Michaelis constants for (all-E)-geranylgeraniol and ATP are 27 nM and 650 microM, respectively.

1-Butanol↗
PubMed1996

[A case of neonatal herpes infection which proved to be a mixed infection of herpes simplex virus type 1 and type 2].

We encountered a case which proved to be a mixed infection of herpes simplex virus (HSV) type 1 and type 2 in retrospective terms by in situ hybridization (ISH) and polymerase chain reaction (PCR). The case was a male. The gestational age was 39 weeks and 2 days. The birth body weight was 3024 g. A fever developed from the age of 6 days and he was admitted to the neonatal intensive care unit at the age of eight days. AST was 1042 IU/L, and ALT 206 IU/L. In spite of treatment, the patient died at the age of 12 days. Using paraffin embedded tissues, we performed the ISH and PCR on the cerebrum, lungs, liver, spleen, bone marrow, adrenal gland, and kidneys. With the ISH, the lungs, liver, spleen and adrenal gland were both HSV type 1 and type 2. With the PCR, only the liver was positive for type 1, and the lungs, liver, spleen, and adrenal gland were positive for type 2. In the ISH, a probe showing a cross reaction between type 1 and type 2 was used for type 1 probe this time. But a type 2 probe and PCR did not show a cross reaction. We concluded that this case confirmed the presence of mixed infection (HSV type 1 and type 2) in neonatal HSV infection.

Herpes Simplex↗
PubMed1995

[Effects of chronic ethanol intake on sugar chain structures of IgG in mice].

IgG is an N-glycosylated glycoprotein, and is secreted from B lymphocyte. Transferrin is also an N-glycosylated glycoprotein secreted from hepatic cell. We have investigated, by lectin-ELISA method, reactivities of sugar chain structures, which are Gal (beta 1, 4) GlcNAc, Fuc (alpha 1, 3 or 6) Gal, NeuAc (alpha 2, 6) Gal, galactose, poly mannoses and NeuAc (alpha 2, 3) Gal, in IgG or transferrin of ddY mice that were given ethanol solution for 3 weeks. In the investigation, we have found that the all reactivities of sugar chain structures in IgG and transferrin remarkably reduced. We have also found that Lewis-X (Gal (beta 1, 4) GlcNAc (3, 1 alpha Fuc)-), which is functional sugar chain structure in the IgG, was markedly reduced with dose-dependent manner. These findings indicate that ethanol decreases activities of glycotransferases in some cells which produce and secrete N-glycosylated glycoproteins. It has been reported that human erythrocyte has also N-glycosylated glycoproteins in the membrane, and its sialic acid and galactose are reduced after chronic ethanol intake. The past and present findings indicate that sugar chain structures of N-glycosylated glycoproteins in both serum and cell membranes are reduced after chronic ethanol intake. Though functions of sugar chain structures of N-glycosylated glycoproteins remain unclear, if functional structures like Lewis-X exist in N-glycosylated glycoproteins, the change of the sugar chain structures may have some relations to diseases occurring after chronic ethanol intake.

Alcoholism↗
PubMed1994

Archaebacterial ether-linked lipid biosynthetic gene. Expression cloning, sequencing, and characterization of geranylgeranyl-diphosphate synthase.

Archaebacterial Sulfolobus acidocaldarius geranylgeranyl-diphosphate (GGPP) synthase (EC 2.5.1.29) catalyzes consecutive condensations of isopentenyl diphosphate with allylic diphosphates to produce GGPP which is the important precursor of archaebacterial ether-linked lipids. We developed an expression screening method for cloning the GGPP synthase gene, which utilizes the carotenoid biosynthesis genes of Erwinia uredovora to visualize a clone expressing GGPP synthase, and then screened a genomic DNA library from S. acidocaldarius for the GGPP synthase gene by using this method. Positive clones were shown to contain GGPP synthase gene by the use of an in vitro assay. Extracts from Escherichia coli transformants catalyzed the condensation of isopentenyl diphosphate with farnesyl diphosphate (FPP) to produce (all-E)-GGPP. The nucleotide sequence of the 2.3-kilobase HindIII fragment of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2. ORF-1 encodes GGPP synthase with the expected molecular weight of 36,873, and ORF-2 encodes a protein with homology for UDP-N-acetylglucosaminedolichyl phosphate N-acetylglucosaminephosphotransferase. The cloned GGPP synthase was partially purified with several chromatographies after heat treatment of cell free extract. This enzyme is extremely thermostable and has an optimal pH at 5.8. Dimethylallyl diphosphate, geranyl diphosphate, and (all-E)-FPP are, in decreasing order of activity, acceptable as allylic substrates to produce (all-E)-GGPP. When dimethylallyl diphosphate or geranyl diphosphate are the allylic substrates, a significant amount of mixture of the products is shorter than GGPP. (2Z,6E)-FPP is not a substrate. This enzyme recognizes the E-configuration of allylic substrate.

Alkyl and Aryl Transferases↗
PubMed1994

[A role for lipoproteins in the recovery from CPPD crystal-induced arthritis].

Calcium pyrophosphate dihydrate (CPPD) crystal-induced inflammation subsides spontaneously, and there have been few reports on its mechanism. It has recently been proposed that lipoproteins in the inflammatory synovial fluid played a role in the recovery from crystal-induced arthritis. This study investigated the inhibitory effect of lipoprotein on the inflammation-inducing activity of CPPD crystals in vitro and in vivo. Lipoproteins (very low density lipoprotein--VLDL, and low density lipoprotein-LDL) were isolated from human serum (in vitro) and rat serum (in vivo) by the sequential ultracentrifugal technique. In the in vitro studies, saline with lipoproteins was mixed with CPPD crystals, and co-incubated with isolated polymorphonuclear leukocytes (PMN). The production of superoxide anion (O-(2)) was measured as an index of inflammatory activity. In the in vivo studies, suspensions of CPPD crystals and rat lipoprotein solutions, were injected into the rat air pouch. The total number of white blood cells (WBC), crystal-containing WBCs, and concentrations of beta-glucronidase and prostaglandin (PG) E2, were measured. In the in vitro studies, O-(2) production by PMNs was more significantly suppressed after treating the CPPD crystals with VLDL or LDL than when the crystals were not treated with VLDL or LDL. In the in vivo studies, treatment of the CPPD crystals with lipoproteins significantly decreased the number of WBC and crystal-containing WBC, as well as the concentration of beta-glucronidase, and tended to depress the concentration of PGE2. These findings suggested that lipoproteins, especially LDL in inflammatory synovial fluid played an important role in the recovery from CPPD crystal-induced arthritis.

Animals↗
PubMed1993

Alteration of the product specificities of prenyltransferases by metal ions.

Effect of several kinds of metal ions on the chain-length distribution of reaction products of polyprenyl diphosphate synthases was investigated. In the presence of Co2+ or Mn2+ octaprenyl-, solanesyl- and decaprenyl diphosphate synthases gave a variety of polyprenyl products with the longest chains being shifted by one or two isoprene units longer than those of the products formed in the presence of Mg2+. Thus octaprenyl diphosphate synthase became to give solanesyl (C45) and decaprenyl (C50) diphosphates when Mg2+ is replaced with Co2+ or Mn2+. Similarly solanesyl diphosphate synthase produced C50- and C55- diphosphates in the presence of Co2+ or Mn2+, and decaprenyl diphosphate synthase gave C55- diphosphate as the longest product in the presence of Mn2+. More remarkable effects were observed with Mn2+ than with Co2+.

Cations, Divalent↗