The effects of epinephrine and electrical stimulation on the activation of glycogen phosphorylase were studied in isolated rat diaphragm and frog sartorius, and rat gastrocnemius in vivo. The resting ratio of phosphorylase, as expressed by the ratio of phosphorylase a to total phosphorylase (-AMP/+AMP ratio) was found to be low of the order of 0.05 in diaphragm and sartorius. In rat gastrocnemius this value was high at 0.26. As measured by the ratio of activity at pH 6.8 to that at pH 8.2 (pH 6.8/8.2 ratio), phosphorylase kinase was essentially in its inactive form under conditions of no stimulation. On treatment with epinephrine, phosphorylase was activated, and there was a significant increase in the activity ratio for phosphorylase kinase, up to 10-fold, indicating that conversion of nonactivated kinase to its activated form had occurred. Epinephrine also produced marked increases, up to 15-fold, in the tissue levels of adenosine 31, 51-monophosphate (cyclic AMP). When muscle was induced to contract by electrical stimulation, phosphorylase was markedly and rapidly activated. In contrast to the effect of epinephrine, electrical stimulation produced no conversion of phosphorylase kinase to its activated form as measured by the pH 6.8/8.2 ratio. This was found for both direct and neural stimulation, at various frequencies, for different times of stimulation, in vitro and in vivo, and in two species. No increase in the tissue levels of cyclic AMP were detected on electrical stimulation. It was concluded that the mechanism of activation of phosphorylase during electrical stimulation is basically different from that produced by adrenergic amines. The data strongly suggests that during muscle contraction phosphorylase is activated by a mechanism which does not involve conversion of phosphorylase kinase to its activated form. In further work, the relationship between phosphorylase activation and muscle contraction was studied. It was found that for any given frequency of stimulation, phosphorylase was activated within 2 sec to a particular ratio for that frequency. On further stimulation, the ratio did not increase. When the temperature was lowered, the steady state phosphorylase ratio for a given frequency was lowered, but activation still occurred rapidly. In experiments in which calcium was removed from the medium by using chelating agents, a correlation was demonstrated between phosphorylase activation and contractile tension. From these results it appears that the mechanism of phosphorylase activation is closely coupled to the contractile mechanism. It is proposed that calcium ion, which is important in excitation-contraction coupling and tension development, is responsible for phosphorylase activation. It is further suggested that calcium ion released into the myoplasm may act with nonactivated phosphorylase kinase to catalyse the conversion of phosphorylase b to phosphorylase a. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

No description available.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-16Bitstream added on 2014-06-13T20:30:22Z : No. of bitstreams: 1 dourado_gkzs_me_arafcf.pdf: 2109934 bytes, checksum: 79ddf2b14ea1fc3f4779c4b22b8e7f81 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Tem sido mostrado que a hesperidina, um flavonóide encontrado nas frutas cítricas e especialmente no suco de laranja, apresenta atividade antiinflamatória e imunomodulatória, entre outras. O presente estudo avaliou os efeitos da ingestão do suco de laranja (SL) como fonte natural da hesperidina, da glicosil hesperidina isolada (GH) e da mistura de ambos (SL-GH), sobre o sistema imune inato de camundongos. Para tanto, foram medidos os parâmetros de produção e inibição da produção de óxido nítrico (NO), e de liberação das citocinas IL-10, IL-12 e TNF- em culturas de macrófagos peritoneais ex vivo. Os macrófagos foram obtidos de grupos de animais previamente tratados com GH, SL, SL-GH ou solução salina (SS). Em seguida as células foram tratadas com GH e lipopolissacarídeo bacteriano (LPS) in vitro. Os resultados mostraram que o tratamento com suco de laranja estimulou a produção de NO de forma moderada (60μmol de NO- 2) pelos macrófagos, aumentou a liberação da citocina IL-10 e diminuiu significativamente a produção de TNF- . O tratamento com G-hesperidina inibiu completamente a produção de NO em macrófagos mesmo na presença de LPS, aumentou a produção da citocina IL-12 e diminuiu a produção de IL-10. Estes resultados sugerem que a ingestão de suco de laranja é benéfica ao sistema imune, podendo apresentar ação contra possíveis agentes infecciosos. A flavanona Ghesperidina isolada pode ser considerada um composto anti-inflamatório nos casos onde ocorre a intensa liberação de óxido nítrico, tais como as doenças inflamatórias / It has been shown that hesperidin, a flavonoid found in citrus fruits and especially in the orange juice, has anti-inflammatory and immunomodulatory, among others. This study evaluated the effects of ingestion of orange juice (SL) as a natural source of hesperidin, isolated glucosyl hesperidin (GH) and a mixture of both (SL-GH) on the innate immune system of mice. To this end, it was measured the parameters of production and inhibiting the production of nitric oxide (NO), and release of cytokines IL-10, IL-12 and TNF-α in cultures of peritoneal macrophages ex vivo. Macrophages were obtained from groups of animals previously treated with GH, SL, SL-GH or saline solution. Then the cells were treated with GH and bacterial lipopolysaccharide (LPS) in vitro. The results showed that treatment with orange juice stimulated the production of NO in a moderate (60µmol NO- 2) by macrophages, increased the release of cytokine IL-10 and significantly decreased the production of TNF-α. Treatment with G-hesperidin completely inhibited NO production in macrophages, even in the presence of LPS, increased the production of cytokine IL-12 and decreased production of IL-10. These results suggest that ingestion of orange juice is beneficial to the immune system, may bring suit against possible infectious agents. The isolated G-flavanone hesperidin can be considered an anti- inflammatory compound in cases where it occurs intense release of nitric oxide, such as inflammatory diseases

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

Orientador: Thais Borges César / Banca: Iracilda Zeppone Carlos / Banca: Telma Maria Braga Costa / Resumo: Tem sido mostrado que a hesperidina, um flavonóide encontrado nas frutas cítricas e especialmente no suco de laranja, apresenta atividade antiinflamatória e imunomodulatória, entre outras. O presente estudo avaliou os efeitos da ingestão do suco de laranja (SL) como fonte natural da hesperidina, da glicosil hesperidina isolada (GH) e da mistura de ambos (SL-GH), sobre o sistema imune inato de camundongos. Para tanto, foram medidos os parâmetros de produção e inibição da produção de óxido nítrico (NO), e de liberação das citocinas IL-10, IL-12 e TNF- em culturas de macrófagos peritoneais ex vivo. Os macrófagos foram obtidos de grupos de animais previamente tratados com GH, SL, SL-GH ou solução salina (SS). Em seguida as células foram tratadas com GH e lipopolissacarídeo bacteriano (LPS) in vitro. Os resultados mostraram que o tratamento com suco de laranja estimulou a produção de NO de forma moderada (60μmol de NO- 2) pelos macrófagos, aumentou a liberação da citocina IL-10 e diminuiu significativamente a produção de TNF- . O tratamento com G-hesperidina inibiu completamente a produção de NO em macrófagos mesmo na presença de LPS, aumentou a produção da citocina IL-12 e diminuiu a produção de IL-10. Estes resultados sugerem que a ingestão de suco de laranja é benéfica ao sistema imune, podendo apresentar ação contra possíveis agentes infecciosos. A flavanona Ghesperidina isolada pode ser considerada um composto anti-inflamatório nos casos onde ocorre a intensa liberação de óxido nítrico, tais como as doenças inflamatórias / Abstract: It has been shown that hesperidin, a flavonoid found in citrus fruits and especially in the orange juice, has anti-inflammatory and immunomodulatory, among others. This study evaluated the effects of ingestion of orange juice (SL) as a natural source of hesperidin, isolated glucosyl hesperidin (GH) and a mixture of both (SL-GH) on the innate immune system of mice. To this end, it was measured the parameters of production and inhibiting the production of nitric oxide (NO), and release of cytokines IL-10, IL-12 and TNF-α in cultures of peritoneal macrophages ex vivo. Macrophages were obtained from groups of animals previously treated with GH, SL, SL-GH or saline solution. Then the cells were treated with GH and bacterial lipopolysaccharide (LPS) in vitro. The results showed that treatment with orange juice stimulated the production of NO in a moderate (60µmol NO- 2) by macrophages, increased the release of cytokine IL-10 and significantly decreased the production of TNF-α. Treatment with G-hesperidin completely inhibited NO production in macrophages, even in the presence of LPS, increased the production of cytokine IL-12 and decreased production of IL-10. These results suggest that ingestion of orange juice is beneficial to the immune system, may bring suit against possible infectious agents. The isolated G-flavanone hesperidin can be considered an anti- inflammatory compound in cases where it occurs intense release of nitric oxide, such as inflammatory diseases / Mestre

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.