Public Library of Science Acta Medica Okayama 1932-6203 19 10 2024 Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6 e0310962 EN Sosuke Munetomo Department of Public Health, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Jumpei Uchiyama Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Iyo Takemura-Uchiyama Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Thamonwan Wanganuttara Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Yumiko Yamamoto Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Toshihiro Tsukui Nippon Zenyaku Kogyo Co. Ltd. Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital Shuji Kanamaru School of Life Science and Technology, Tokyo Institute of Technology Hideyuki Kanda Department of Public Health, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Osamu Matsushita Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Methicillin-resistant Staphylococcus spp. present challenges in clinical and veterinary settings because effective antimicrobial agents are limited. Phage-encoded peptidoglycan-degrading enzyme, endolysin, is expected to be a novel antimicrobial agent. The enzymatic activity has recently been shown to be influenced by the linker between functional domains in the enzyme. S6_ORF93 (ORF93) is one of the endolysins derived from previously isolated Staphylococcus giant phage S6. The ORF93 was speculated to have a catalytic and peptidoglycan-binding domain with a long linker. In this study, we examined the influence of linker shortening on the characteristics of ORF93. We produce wild-type ORF93 and the linker deletion mutants using an Escherichia coli expression system. These mutants were designated as ORF93-Delta 05, ORF93-Delta 10, ORF93-Delta 15, and ORF93-Delta 20, from which 5, 10, 15, and 20 amino acids were removed from the linker, respectively. Except for the ORF93-Delta 20, ORF93 and its mutants were expressed as soluble proteins. Moreover, ORF93-Delta 15 showed the highest yield and bacteriolytic activity, while the antimicrobial spectrum was homologous. The cold storage experiment showed a slight effect by the linker deletion. According to our results and other studies, linker investigations are crucial in endolysin development. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 67 1 2013 Phospholipase C Produced by Clostridium botulinum Types C and D:Comparison of Gene, Enzymatic, and Biological Activities with Those of Clostridium perfringens Alpha-toxin 9 18 EN Ni Nengah Dwi Fatmawati Yoshihiko Sakaguchi Tomonori Suzuki Masataka Oda Kenta Shimizu Yumiko Yamamoto Jun Sakurai Osamu Matsushita Keiji Oguma Original Article 10.18926/AMO/49252 Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 66 4 2012 Intraprostatic Botulinum Neurotoxin Type A Injection for Benign Prostatic Hyperplasia:Preliminary Results with a Newly Purified Neurotoxin 291 297 EN Teruhiko Yokoyama Yumiko Yamamoto Tomonori Suzuki Keiji Oguma Atsushi Nagai Original Article 10.18926/AMO/48668 Several studies have demonstrated the efficacy of intraprostatic injection of botulinum neurotoxin type A (BoNT/A) against symptomatic benign prostatic hyperplasia (BPH). The most commonly used BoNT/A product, Botox®, forms large complexes and composed of neurotoxin (NTX) as well as non-toxic components. We purified NTX lacking non-toxic components. We investigated the efficacy of this newly purified NTX for men with BPH. Ten male patients (mean age, 70.0 years) with BPH received 100 units (prostate volume [PV] ＜30ml) or 200 units (PV ｧ30ml) of NTX injected into the prostate via a minimally invasive outpatient technique. Evaluation included uroflowmetry, postvoid residual urine volume (PVR), PV, and International Prostate Symptom Score (IPSS) measured at baseline and 1, 3, 6, and 12 months post-treatment. The status of 7 of the 10 patients examined was found to have improved within 1 month of treatment. The mean IPSS decreased from 23.8±7.0 to 16.3±10.3 (p＝0.0093) at 1 month, to 14.9±8.2 (p＝0.0074) at 3 months, and to 16.9±7.3 (p＝0.018) at 12 months. The mean PV decreased from 47.8±21.2 to 39.2±19.5ml (p＝0.0076) at 3 months. The PVR improved at 3 and 6 months post-treatment. Intraprostatic NTX injection induces prostate shrinkage and is effective in men with BPH. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 66 3 2012 Clostridium botulinum Type E Toxins Bind to Caco-2 Cells by a Different Mechanism from That of Type A Toxins 253 261 EN Kai Zhang Yumiko Yamamoto Tomonori Suzuki Kenji Yokota Shaobo Ma Ni Nengah Dwi Fatmawati Keiji Oguma Original Article 10.18926/AMO/48565 Cultured Clostridium botulinum strains produce progenitor toxins designated as 12S, 16S, and 19S toxins. The 12S toxin consists of a neurotoxin (NTX, 7S) and a non-toxic non-hemagglutinin (NTNH). The 16S and 19S toxins are formed by conjugation of the 12S toxin with hemagglutinin (HA), and the 19S toxin is a dimer of the 16S toxin. Type A cultures produce all 3 of these progenitor toxins, while type E produces only the 12S toxin. The 7S toxin is cleaved into heavy (H) and light (L) chains by a protease(s) in some strains, and the H chain has 2 domains, the N-terminus (Hn) and C-terminus (Hc). It has been reported that type A toxins bind to the intestinal cells or cultured cells via either HA or Hc. In this study, we investigated the binding of type A and E toxins to Caco-2 cells using Western blot analysis. Both the type E 7S and 12S toxins bound to the cells, with the 7S toxin binding more strongly, whereas, in the type A strain, only the 16S/19S toxins showed obvious binding. Pre-incubation of the type E 7S toxin with IgG against recombinant type E Hc significantly inhibited the 7S toxin binding, indicating that Hc might be a main binding domain of the type E toxin. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2001 Carboxyl­Terminal Processing of Precursor D1 Protein of Photosystem II Reaction Center EN No potential conflict of interest relevant to this article was reported.