Kazuhisa Murakami

Publications (10)23.36 Total impact

• Article: [Identification of novel therapeutically effective antibiotics using silkworm infection model].

  Hiroshi Hamamoto, Makoto Urai, Atmika Paudel, Ryo Horie, Kazuhisa Murakami, Kazuhisa Sekimizu
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  ABSTRACT: Most antibiotics obtained by in vitro screening with antibacterial activity have inappropriate properties as medicines due to their toxicity and pharmacodynamics in animal bodies. Thus, evaluation of the therapeutic effects of these samples using animal models is essential in the crude stage. Mammals are not suitable for therapeutic evaluation of a large number of samples due to high costs and ethical issues. We propose the use of silkworms (Bombyx mori) as model animals for screening therapeutically effective antibiotics. Silkworms are infected by various pathogenic bacteria and are effectively treated with similar ED(50) values of clinically used antibiotics. Furthermore, the drug metabolism pathways, such as cytochrome P450 and conjugation systems, are similar between silkworms and mammals. Silkworms have many advantages compared with other infection models, such as their 1) low cost, 2) few associated ethical problems, 3) adequate body size for easily handling, and 4) easier separation of organs and hemolymph. These features of the silkworm allow for efficient screening of therapeutically effective antibiotics. In this review, we discuss the advantages of the silkworm model in the early stages of drug development and the screening results of some antibiotics using the silkworm infection model.
  YAKUGAKU ZASSHI 01/2012; 132(1):79-84. · 0.37 Impact Factor
• Article: Evaluation of target specificity of antibacterial agents using Staphylococcus aureus ddlA mutants and D-cycloserine in a silkworm infection model.

  Kenji Kurokawa, Hiroshi Hamamoto, Miki Matsuo, Satoshi Nishida, Noriko Yamane, Bok Luel Lee, Kazuhisa Murakami, Hideki Maki, Kazuhisa Sekimizu
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  ABSTRACT: The availability of a silkworm larva infection model to evaluate the therapeutic effectiveness of antibiotics was examined. The 50% effective doses (ED50) of D-cycloserine against the Staphylococcus aureus ddlA mutant-mediated killing of larvae were remarkably lower than those against the parental strain-mediated killing of larvae. Changes in MICs and ED50 of other antibiotics were negligible, suggesting that these alterations are d-cycloserine selective. Therefore, this model is useful for selecting desired compounds based on their therapeutic effectiveness during antibiotic development.
  Antimicrobial Agents and Chemotherapy 07/2009; 53(9):4025-7. · 4.84 Impact Factor
• Article: Staphylococcus aureus MurC participates in L-alanine recognition via histidine 343, a conserved motif in the shallow hydrophobic pocket.

  Kenji Kurokawa, Satoshi Nishida, Mihoko Ishibashi, Hikaru Mizumura, Kohji Ueno, Takashi Yutsudo, Hideki Maki, Kazuhisa Murakami, Kazuhisa Sekimizu
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  ABSTRACT: UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.
  Journal of Biochemistry 04/2008; 143(3):417-24. · 2.37 Impact Factor
• Article: Staphylococcus aureus clinical isolate with high-level methicillin resistance with an lytH mutation caused by IS1182 insertion.

  Takaji Fujimura, Kazuhisa Murakami
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  ABSTRACT: We previously reported that deficiency of the lytH gene, whose product is homologous to lytic enzymes, caused the elevation of methicillin resistance in Staphylococcus aureus strain SR17238, a strain of S. aureus with a low level of resistance to methicillin (low-level MRSA) (J. Bacteriol. 179:6294-6301, 1997). In this study, we demonstrated that deficiency of lytH caused the same phenomenon in four other clinical isolates of low-level MRSA, suggesting this deficiency to exist in clinical isolates. We therefore searched the region including lytH in 127 clinical isolates of MRSA by PCR and found one strain, SR17164 (methicillin MIC, 1,600 microg/ml), in which the lytH gene was inactivated by insertion sequence IS1182. lytH::IS1182 was replaced with intact lytH in this strain by integration and excision of the plasmid carrying the lytH region. Recombinants with intact lytH genes showed methicillin MICs of 800 microg/ml, twofold lower than those of the recombinants with lytH::IS1182 and the parent. In addition, S. aureus SR17164, which has a high level of methicillin resistance, had properties similar to those caused by lytH deficiency; that is, the resistance levels of strain SR17164 and lytH-deficient variants from strain SR17238 were not significantly affected by llm inactivation, which greatly lowered resistance levels in most other high-level MRSA strains. These findings suggest that lytH inactivation contributed, to some extent, to the resistance level of S. aureus SR17164. To the best of our knowledge, this strain is the first clinical isolate of MRSA for which the genetic base for high-level resistance has been clarified.
  Antimicrobial Agents and Chemotherapy 03/2008; 52(2):643-7. · 4.84 Impact Factor
• Article: Effects of molecular mass and hydrophobicity on transport rates through non-specific pathways of the silkworm larva midgut.

  Hiroshi Hamamoto, Koushirou Kamura, Iony Manitra Razanajatovo, Kazuhisa Murakami, Tomofumi Santa, Kazuhisa Sekimizu
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  ABSTRACT: We previously reported that therapeutic drug effects in the silkworm infection model are largely influenced by midgut permeability. In this report, we describe the effects of drug molecular mass and hydrophobicity on transport through the silkworm larva midgut membrane. Hydrophilic compounds with a molecular mass of greater than 400Da did not permeate the silkworm larva midgut, and the hydrophobicity of similar-sized compounds had positive effects on the transport rate. Furthermore, we compared transport rates through the midgut membrane between cefcapene sodium (CFPN-Na) and cefcapene pivoxil (CFPN-PI), which is a CFPN-Na prodrug. The in vitro transport rate of CFPN-PI was three times faster than that of CFPN-Na. Moreover, when CFPN-PI and CFPN-Na were injected into the living silkworm larva midgut, CFPN-PI appeared rapidly in the haemolymph, whereas CFPN-Na did not. The 50% effective dose (ED50) of CFPN-PI administered via the midgut was one-sixth that of CFPN-Na. These findings suggest that the general features of the non-specific transport route are similar between silkworm larvae and mammals.
  International Journal of Antimicrobial Agents 08/2005; 26(1):38-42. · 4.13 Impact Factor
• Article: Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus.

  Miki Matsuo, Kenji Kurokawa, Satoshi Nishida, Yan Li, Haruto Takimura, Chikara Kaito, Norio Fukuhara, Hideki Maki, Kenji Miura, Kazuhisa Murakami, Kazuhisa Sekimizu
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  ABSTRACT: The murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the murB gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity.
  FEMS Microbiology Letters 06/2003; 222(1):107-13. · 2.04 Impact Factor
• Article: A model for antiHelicobacter pylori chemotherapy using an experimental murine infection withHelicobacter felis

  Yoshinao Kobayashi, Masayoshi Doi, Hiroshi Nagata, Kazunori Sasaki, Toshiyuki Maruyama, Kazuhisa Murakami
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  ABSTRACT: Colonization ofHelicobacter felis was observed to continue for at least 24 weeks in a BALB/c mouse stomach after oral inoculation of the bacteria. Bacteria were observed on the surface of the gastric mucosa and in the gastric pits as is typically seen withHelicobacter pylori infection in the human stomach.H. felis-colonized mice were treated for 5 consecutive days with various chemotherapeutic regimens used againstH. pylori for humans. The status ofH. felis infection was then assessed by the urease test 2 weeks after the final treatment. The clearance rates were 100% for a triple combination of amoxicillin (15 mg/kg), metronidazole (10 mg/kg) and bismuth subnitrate (5 mg/kg), 71% for amoxicillin alone (15 mg/kg), 80% for metronidazole alone (15 mg/kg), and 0% for bismuth subnitrate (60 mg/kg) or omeprazole (60 mg/kg) alone. Bacterial recurrence occurred more often in mice treated with amoxicillin alone than with the triple regimen during the 24-week posttreatment period. These results were very similar to the efficacy of each regimen obtained in clinical trials. Thus, this mouse model usingH. felis may be useful for evaluating drugs or regimens for the treatment of humanH. pylori infections.
  Journal of Infection and Chemotherapy 01/1998; 4(4):195-200. · 1.80 Impact Factor
• Article: In vitro antibacterial activity of a new oral cephalosporin S-1090

  Yoshihide Komatsu, Kazuhisa Murakami, Hiroshi Nagata, Kiyoshi Motokawa, Masayoshi Doi, Isao Higashiyama, Shimaru Sasaki, Tadashi Yoshida, Shogo Kuwahara
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  ABSTRACT: A new oral cephem antibiotic S-1090 was evaluated for its in vitro antibacterial activity in comparison with cefdinir, cefpodoxime, and cefaclor. S-1090 had fairly potent antibacterial activity when compared with cefdinir, cefpodoxime, and cefaclor. At concentrations below 0.78 μg/mL, S-1090 inhibited 90% of the strains of methicillin-susceptibleStaphylococcus aureus, most streptococci,Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Neisseria gonorrhoeae, andHaemophilus influenzae. S-1090 was minimally active against methicillin-resistantS. aureus, enterococci,Serratia marcescens, Citrobacter freundii, Enterobacter aerogenes, andEnterobacter cloacae, and inactive againstPseudomonas aeruginosa and Xanthomonas maltophilia. S-1090 was stable to hydrolysis by various penicillinases and cephalosporinases, but was rapidly hydrolyzed by penicillinase produced byMoraxella (Branhamella) catarrhalis, by plasmid-mediated OXA-1 type penicillinase, and by cephalosporinases produced byProteus vulgaris andBacteroides fragilis. S-1090 had high affinities for the “lethal target PBPs” ofS. aureus andE. coli, and showed potent bactericidal activity against these bacterial species.
  Journal of Infection and Chemotherapy 02/1997; 3(1):38-48. · 1.80 Impact Factor
• Article: The 7{alpha}-methoxy substituent in cephem or oxacephem antibiotics enhances in vivo anti-Helicobacter felis activity in mice after oral administration

  Yoshinao Kobayashi, Masayoshi Doi, Hiroshi Nagata, Tadatoshi Kubota, Masaharu Kume, Kazuhisa Murakami
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  ABSTRACT: The in vivo antibacterial activities of 7α-methoxy-cephems or 7α-methoxy-1-oxacephems and their demethoxy congeners after oral administration were compared in a mouse model of gastric infection with Helicobacter felis . The MICs of all four pairs of compounds with and without the 7α-methoxy substituent were within one two-fold dilution for H. felis and Helicobacter pylori , but the 7α-methoxy compounds were at least four-fold more active at bacterial eradication than their demethoxy congeners. Since these compounds are not absorbed after oral administration, they are likely to have gained direct access to the bacteria from the gastric cavity. Thus the 7α-methoxy substituent may enhance in vivo activity by promoting the gastric mucus permeability of the compounds or by allowing the compounds to remain longer in the gastric cavity.
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  Available from: iiarjournals.org

  Article: Pharmacokinetics and tissue penetration of a new carbapenem, doripenem, intravenously administered to laboratory animals.

  Toshihiko Hori, Masao Nakano, Yasuo Kimura, Kazuhisa Murakami
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  ABSTRACT: The pharmacokinetic properties of doripenem following 20 mg/kg i.v. infusion were studied in various laboratory animals. The concentrations of doripenem in plasma, urine and tissue samples were determined by bioassay. Mean AUC0(0-infinityS) (microg x h/ml) and urinary recoveries (UR, %, 0-24 h) were 14.1 and 36.3 in mice, 9.3 and 42.1 in rats, 47.9 and 47.6 in rabbits, 78.6 and 83.1 in dogs and 44.1 and 51.0 in monkeys, respectively. In monkeys, with co-administration of probenecid, the mean AUC(0-infinity) of doripenem increased about 2.2 times and urinary excretion was delayed slightly. In mice, the doripenem level was highest in the blood plasma, followed by the kidney, liver, lung, heart and spleen. These doripenem levels in various tissues rapidly decreased and no accumulation was observed. Serum protein binding rates (%) of doripenem were 25.2 in mice, 35.2 in rats, 11.8 in rabbits, 10.2 in dogs, 6.1 in monkeys and 8.1 in humans, respectively.
  In vivo (Athens, Greece) 20(1):91-6. · 1.17 Impact Factor