RESUMO
We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.
Assuntos
Anticorpos Catalíticos/uso terapêutico , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Catalíticos/genética , Biópsia , Infecções por Helicobacter/patologia , Helicobacter pylori/enzimologia , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/uso terapêutico , Cadeias kappa de Imunoglobulina/uso terapêutico , Camundongos , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , RNA Mensageiro/genética , Gastropatias/imunologia , Gastropatias/microbiologia , Gastropatias/patologia , Urease/química , Urease/genéticaRESUMO
In the course of research on the bioactive constituents of woody plants in the Cyugoku area of Japan, a methanol extract of the leaves of Alnus japonica were found to have strong antioxidative activity. Ethyl acetate soluble and n-buthanol soluble fractions of the methanol extract had a potent antioxidative effect. Both fractions were purified by silica gel column chromatography and HPLC using an ODS column to give four new diarylheptanoids along with known diarylheptanoids and flavonoids. These new compounds were elucidated to be 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-(4-hydroxyphenyl)-3-heptanone-5-O-beta-D-xylopyranoside (1), 1-(3,4-dihydroxyphenyl)-5-hydroxy-7-(4-hydroxyphenyl)-3-heptanone-5-O-beta-D-xylopyranoside (2), 1,7-bis-(3,4-dihydroxyphenyl)-5-hydroxy-3-heptanone-5-O-[2-(2-methylbutenoyl)]-beta-D-xylopyranoside (3) and 1,7-bis-(3,4-dihydroxyphenyl)-5-methoxy-3-heptanone (4) using spectral methods and especially 1H-, 13C-NMR and 2D-NMR measurements. The isolated compounds including their main constituent, oregonin (5), were tested for antioxidative activity. Some of these compounds having two catechol structures showed potent antioxidative activity. Compounds having one catechol structure showed moderate antioxidative activity, but a peracetate of 5 having no catechol structure exhibited no antioxidative activity. Thus the catechol structure of the diarylheptanoids is indispensable for antioxidative activity.
Assuntos
Alnus/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Diarileptanoides/química , 1-Butanol , Acetatos , Compostos de Bifenilo , Cromatografia Líquida de Alta Pressão , Diarileptanoides/isolamento & purificação , Etanol , Flavonas/química , Flavonas/isolamento & purificação , Sequestradores de Radicais Livres/química , Espectroscopia de Ressonância Magnética , Metanol , Oxidantes/química , Oxirredução , Picratos/química , Extratos Vegetais/química , Folhas de Planta/química , Solventes , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Superóxidos/químicaRESUMO
Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria have long been sought for potential therapeutic use. Helicobacter pylori urease plays a crucial role for the survival of this bacterium in the highly acidic conditions of human stomach. The HpU-9 monoclonal antibody (mAb) raised against H. pylori urease recognized the alpha-subunit of the urease, but only slightly recognized the beta-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the beta-subunit. The cleavage reaction catalyzed by HpU-9 light chain (HpU-9-L) followed the Michaelis-Menten equation with a K(m) of 1.6 x 10(-5) m and a k(cat) of 0.11 min(-1), suggesting that the cleavage reaction was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L efficiently cleaved the beta-subunit but not the alpha-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the beta-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the digestion by HpU-9-L was specific. In summary, we succeeded in the preparation of a catalytic antibody light chain capable of specifically digesting the beta-subunit of H. pylori urease.
