RESUMEN
The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-beta-D-mannuronate blocks, leading to the release of O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1-->4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligomannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligomannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G-M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G-G nor the M-G diads. This lyase should therefore be referred to as a mannuronate beta-eliminase, indicating that the enzyme performs beta-elimination on mannuronate residues only, from both the M-M and G-M diads of alginates.
Asunto(s)
Alginatos/metabolismo , Moluscos/enzimología , Oligosacáridos/química , Polisacárido Liasas/metabolismo , Ácidos Urónicos/química , Alginatos/aislamiento & purificación , Animales , Sitios de Unión , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Ácidos Hexurónicos/química , Espectroscopía de Resonancia Magnética , Manosa/análogos & derivados , Oligosacáridos/análisis , Oligosacáridos/metabolismo , Concentración Osmolar , Ácidos Urónicos/análisisRESUMEN
The 1H and 13C NMR chemicals shifts of the various saturated and unsaturated timers obtained by acid or enzymatic depolymerisation of homopolymeric blocks of alginates are reported. In addition, 13C NMR chemical shifts are assigned for several saturated oligomers of higher polymerisation degrees. Breakdown of alginate and of homopolymeric alginate blocks by Haliotis tuberculata alginate lyase was monitored with 1H NMR spectroscopy and the signals relevant to the identification of the lyase products are pointed out. The enzymes performs beta-elimination on the mannuronic acid residues, independently of their immediately surrounding neighbours. Application of this approach to the analysis of the substrate specificity of alginate lyases is discussed.
Asunto(s)
Alginatos/química , Moluscos/enzimología , Polisacárido Liasas/metabolismo , Animales , Eucariontes/química , Ácido Glucurónico , Ácidos Hexurónicos/química , Ácidos Hexurónicos/aislamiento & purificación , Hidrólisis , Klebsiella pneumoniae/enzimología , Espectroscopía de Resonancia Magnética , Especificidad por Sustrato , Ácidos Urónicos/química , Ácidos Urónicos/aislamiento & purificaciónRESUMEN
A nucleic acid hybridization assay was used to evaluate inhibitory activity of antiviral compounds against hepatitis A virus (HAV) in cell culture and compared to radioimmunoassay by analysis of variance procedure. The 5' genomic end of the HM-175 strain was used as digoxigenin-labeled RNA probe. Dot-blot examination showed a reduction of detectable HAV RNA in infected cells when treated with amphotericin B. An antiviral dose-effect was shown by statistical analysis of densitometric measures of hybridization signals. Comparison between molecular hybridization assay and radioimmunoassay by analysis of variance procedure showed the equivalence of both methods. Data previously obtained on selected drugs by antigen and infectious titres determinations were confirmed by hybridization assay and make possible digoxigenin-labeled RNA probe use to measure an antiviral dose-effect for screening of hepatitis A antiviral compounds.
Asunto(s)
Anfotericina B/farmacología , Antivirales/farmacología , Digoxigenina/farmacología , Virus de la Hepatitis A Humana/efectos de los fármacos , Hibridación de Ácido Nucleico , Sondas ARN/efectos de los fármacos , Amidas , Carragenina/farmacología , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacología , Ácido Glicirrínico , Técnicas In Vitro , Pirazoles , Radioinmunoensayo , Ribonucleósidos/farmacología , RibosaRESUMEN
Sulphated polysaccharides such as iota-, lambda- and kappa-carrageenans showed a potent inhibitory effect on the replication of hepatitis A virus (HAV) in the human hepatoma cell line PLC/PRF/5. No cytotoxic effects were detected with concentrations of carrageenans up to 200 micrograms/ml. The selectivity indices of these substances, calculated as the ratio of the dose that reduced the number of viable cells to 50% (CD50) to the effective dose that inhibited 50% of viral antigen expression (ED50), were greater than 400 with iota-carrageenan, greater than 222 with lambda-carrageenan and greater than 10 with kappa-carrageenan. The selectivity index of ribavirin (reference substance) was only 5. The 3 types of carrageenans resulted in concentration-dependent reduction of HAV-antigen expression and HAV infectivity. lota-and lambda-carrageenan emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.
Asunto(s)
Carragenina/farmacología , Hepatovirus/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/microbiología , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Regulación Viral de la Expresión Génica , Hepatitis A/tratamiento farmacológico , Hepatovirus/crecimiento & desarrollo , Humanos , Técnicas In Vitro , Ribavirina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
The activity of various antibacterial agents (amoxicillin, josamycin, doxycycline and metronidazole) was established in vitro using a rapid micromethod. The activity of these agents, which are widely used in oral medicine, was evaluated against microorganisms responsible for periodontitis and bucco-dental infections. Their action against alpha-hemolytic streptococci (including pneumococci) which make up the majority of the indigenous oral flora was also tested. Amoxicillin was found to be effective against all the strains tested. Doxycycline was active against periodontal bacteria, but not against 50% of the streptococcal flora. Josamycin was found to be effective against streptococci, but appeared without effect on Eikenella corrodens and Actinobacillus actinomycetemcomitans. Metronidazole, inactive against streptococci, displayed greater activity towards the strict anaerobes. The use of these antibiotics for the treatment of bucco-dental infections, especially periodontitis, is discussed. For periodontitis and periodontal suppurations, antimicrobial agents present a valuable adjunct to local treatments such as scaling or rootplaning. This may prevent more serious infections such as endocarditis that can develop after tooth extraction.