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Antibacterial constituents of Nitzschia longissima
Tenth World Congress
533
phases by adding chloroform-water (1 : 1 v/v). The lipophilic phase was active against three Gram-positive and six Gram-negative bacteria. The lipophilic phase was fractionated on a column into compound groups and the fractions were tested against Bacillus subtilis. The active fraction was analysed in GC-FID and the constituents were identified as undecanoic acid. tetradecanoic acid, hexadecanoic acid (ante iso), hexadecanoic acid, cis 9 hexadecenoic acid and cis 5, 8, 11, 14 eicosatetraeonic acid.
Toxicological and pharmacological principles of Nerium odorum on a few dermatophytes. V. K. MANJULA,A. H1LDA, V.M. RAMESH and S. RAJA RAJAN (C.A.S. in Botany, University of Madras, Guindy Campus, Madras-25, India). KERATINOPHILIC fungi inhabiting the playgrounds of primary schools were isolated and identified. Most of the etiological agents of dermatomycosis were keratinophilic fungi. A children's playground represents a favourable environment for the occurrence of a wide variety of fungi, and they are therefore expected to play an important role in the epidemiology of human mycosis. Many higher plant extracts act as potent inhibitors of fungi. The inhibitory effect of the toxic principle in the flower extract of Nerium odorum on Trichophyton mentagrophytes and Microsporum gypseum is reported. Sensitivity tests in fluid and solid media were assessed; 100% inhibitions of both Trichophyton and Microsporum was seen up to I x 10-5/ag/ml. However, further dilution of the extract indicated that Microsporum was more sensitive than Trichophyton. The efficiency of the toxic principle in Nerium odorum flowers to inhibit the two dermatophytic fungi seemed to pave the way for the control of dermatophytes. Fungitoxic substances in the flowers of Nerium odorum was further detected by a bioautographic technique using Cladosporium herbarum. The efficiency of the toxic zone located in the chromatogram was further assessed by the sensitivity response of Microsporum and Trichophyton. The efficiency of the antifungal substance of Nerium odorum in the control of dermatophytes is discussed.
Purification and characterization of two fibrinolytic enzymes of Bothrops jararaca venom. M. MARUYAMA, M. SUGI~I, H. MIrIARA, A. S. KAMIGUTI and R. D. G. THEAKSTOr~(Department of Physiology, Miyazaki Medical College, Japan). WE PURIFIEDfibrinolytic enzymes from Bothrops jararaca venom for investigating their enzymatic characteristics. Apparently, the venom had three fibrinolytic enzymes and we purified two of them. Purification was performed by two successive steps of Afli-Gel Blue affinity chromatography preceded by Sephadex G-75 (medium size) column chromatography. The final products, FE I and FE II, were obtained by Sephacril S-200 column chromatography or Sephadex G-75 superfine column chromatography. FE I and FE II showed a single protein band by SDS-polyacrylamide gel electrophoresis and the mol. wts were 47,000__+2000 and 21,400__500, respectively. The specific activities of FE I and FE II were calculated using plasmin as a standard, and were found to be 2.04 U/mg protein and 6.34 U/mg protein, respectively. No coagulating activity was detected in FE I and FE II. Because both enzymes showed fibrinolytic activity in the plasminogen free plate method as well as the plasminogen rich plate, they seemed to have no plasminogen activating activity. Two enzymes were strongly inhibited by 1,10-phenanthroline, EDTA and dithiothreitol, and slightly inhibited by soy bean trypsin inhibitor and leupeptin. No inhibition was observed by APMSF, TLCK and Elastatinal. From the above data, both enzymes were thought to be metalloproteases and the S-S bond might be important in expressing their activity.
Anti-tetanus human monoclonal antibodies with high protective activity. M. MATSUDA, M. KAMEi, N. SUOIMOTO, Y. MA and S. Hxsmzur,~ (Department of Tuberculosis Research (Bacterial Toxinology), Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan). THE PURPOSEof this study is to establish hybrid cell lines producing anti-tetanus human monoclonal antibodies (MAbs) practically useful for prevention and therapy of human tetanus. We generated five stable hybrid cell lines producing, in serum-free medium, large amounts of anti-tetanus human MAb with high neutralizing activity, by fusion of peripheral lymphocytes from humans hyperimmunized with tetanus toxoid with mouse/human heteromyelomas SHMD-33 or RF-SI. By using ELISA, we found that these MAbs have high binding affinity (c. 10HM -t) to the toxin and are directed against three different functional domains of the tetanus toxin molecule: one against fragment [A], the N-terminal domain, another against fragment [C], the C-terminal domain, and the remaining three against fragment [B], the middle domain. The individual MAbs, as single reagents, could protect mice completely from death due to tetanus toxin. Appropriate combinations of three or four kinds of MAbs resulted in markedly increased neutralizing activity, on the basis of toxin-specific IgG content, comparable with that of anti-tetanus human polyclonal immunoglobulin preparations currently used clinically. Prevous i.v. injection of MAb could protect mice against challenge with toxin. Mice previously injected with toxin could be rescued by injecting MAb even 10hr after toxin injection. Thus these MAbs will be useful for both the prevention and treatment of clinical tetanus.
533
phases by adding chloroform-water (1 : 1 v/v). The lipophilic phase was active against three Gram-positive and six Gram-negative bacteria. The lipophilic phase was fractionated on a column into compound groups and the fractions were tested against Bacillus subtilis. The active fraction was analysed in GC-FID and the constituents were identified as undecanoic acid. tetradecanoic acid, hexadecanoic acid (ante iso), hexadecanoic acid, cis 9 hexadecenoic acid and cis 5, 8, 11, 14 eicosatetraeonic acid.
Toxicological and pharmacological principles of Nerium odorum on a few dermatophytes. V. K. MANJULA,A. H1LDA, V.M. RAMESH and S. RAJA RAJAN (C.A.S. in Botany, University of Madras, Guindy Campus, Madras-25, India). KERATINOPHILIC fungi inhabiting the playgrounds of primary schools were isolated and identified. Most of the etiological agents of dermatomycosis were keratinophilic fungi. A children's playground represents a favourable environment for the occurrence of a wide variety of fungi, and they are therefore expected to play an important role in the epidemiology of human mycosis. Many higher plant extracts act as potent inhibitors of fungi. The inhibitory effect of the toxic principle in the flower extract of Nerium odorum on Trichophyton mentagrophytes and Microsporum gypseum is reported. Sensitivity tests in fluid and solid media were assessed; 100% inhibitions of both Trichophyton and Microsporum was seen up to I x 10-5/ag/ml. However, further dilution of the extract indicated that Microsporum was more sensitive than Trichophyton. The efficiency of the toxic principle in Nerium odorum flowers to inhibit the two dermatophytic fungi seemed to pave the way for the control of dermatophytes. Fungitoxic substances in the flowers of Nerium odorum was further detected by a bioautographic technique using Cladosporium herbarum. The efficiency of the toxic zone located in the chromatogram was further assessed by the sensitivity response of Microsporum and Trichophyton. The efficiency of the antifungal substance of Nerium odorum in the control of dermatophytes is discussed.
Purification and characterization of two fibrinolytic enzymes of Bothrops jararaca venom. M. MARUYAMA, M. SUGI~I, H. MIrIARA, A. S. KAMIGUTI and R. D. G. THEAKSTOr~(Department of Physiology, Miyazaki Medical College, Japan). WE PURIFIEDfibrinolytic enzymes from Bothrops jararaca venom for investigating their enzymatic characteristics. Apparently, the venom had three fibrinolytic enzymes and we purified two of them. Purification was performed by two successive steps of Afli-Gel Blue affinity chromatography preceded by Sephadex G-75 (medium size) column chromatography. The final products, FE I and FE II, were obtained by Sephacril S-200 column chromatography or Sephadex G-75 superfine column chromatography. FE I and FE II showed a single protein band by SDS-polyacrylamide gel electrophoresis and the mol. wts were 47,000__+2000 and 21,400__500, respectively. The specific activities of FE I and FE II were calculated using plasmin as a standard, and were found to be 2.04 U/mg protein and 6.34 U/mg protein, respectively. No coagulating activity was detected in FE I and FE II. Because both enzymes showed fibrinolytic activity in the plasminogen free plate method as well as the plasminogen rich plate, they seemed to have no plasminogen activating activity. Two enzymes were strongly inhibited by 1,10-phenanthroline, EDTA and dithiothreitol, and slightly inhibited by soy bean trypsin inhibitor and leupeptin. No inhibition was observed by APMSF, TLCK and Elastatinal. From the above data, both enzymes were thought to be metalloproteases and the S-S bond might be important in expressing their activity.
Anti-tetanus human monoclonal antibodies with high protective activity. M. MATSUDA, M. KAMEi, N. SUOIMOTO, Y. MA and S. Hxsmzur,~ (Department of Tuberculosis Research (Bacterial Toxinology), Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan). THE PURPOSEof this study is to establish hybrid cell lines producing anti-tetanus human monoclonal antibodies (MAbs) practically useful for prevention and therapy of human tetanus. We generated five stable hybrid cell lines producing, in serum-free medium, large amounts of anti-tetanus human MAb with high neutralizing activity, by fusion of peripheral lymphocytes from humans hyperimmunized with tetanus toxoid with mouse/human heteromyelomas SHMD-33 or RF-SI. By using ELISA, we found that these MAbs have high binding affinity (c. 10HM -t) to the toxin and are directed against three different functional domains of the tetanus toxin molecule: one against fragment [A], the N-terminal domain, another against fragment [C], the C-terminal domain, and the remaining three against fragment [B], the middle domain. The individual MAbs, as single reagents, could protect mice completely from death due to tetanus toxin. Appropriate combinations of three or four kinds of MAbs resulted in markedly increased neutralizing activity, on the basis of toxin-specific IgG content, comparable with that of anti-tetanus human polyclonal immunoglobulin preparations currently used clinically. Prevous i.v. injection of MAb could protect mice against challenge with toxin. Mice previously injected with toxin could be rescued by injecting MAb even 10hr after toxin injection. Thus these MAbs will be useful for both the prevention and treatment of clinical tetanus.