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1.
Biotechnol Lett ; 26(23): 1803-8, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15672218

RESUMO

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis, was achieved by using more stable vectors in the order of S11 replication origin-containing episomal vector < full-length K. lactis plasmid pKD1-containing vector < centromeric vector < chromosome-integrated vectors. Cells containing a PGK (phosphoglycerate kinase) promoter-driven integration vector grown in non-selective rich medium achieved the highest level of secretion, approximately 100 microg lysozyme secretion ml(-1) culture: this level was approximately 10-fold higher than that achieved by episomal vectors. An additional copy of the protein disulfide isomerase gene further facilitated the secretion.


Assuntos
Kluyveromyces/enzimologia , Kluyveromyces/genética , Muramidase/biossíntese , Muramidase/genética , Engenharia de Proteínas/métodos , Transfecção/métodos , Melhoramento Genético/métodos , Humanos , Muramidase/química , Muramidase/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
2.
Circulation ; 107(3): 477-84, 2003 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-12551874

RESUMO

BACKGROUND: The development of heart failure is tightly correlated with a decrease in the stoichiometric ratio for FKBP12.6 binding to the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR). We report that a new drug, the 1,4-benzothiazepine derivative JTV519, reverses this pathogenic process. JTV519 is known to have a protective effect against Ca2+ overload-induced myocardial injury. METHODS AND RESULTS: Heart failure was produced by 4 weeks of rapid right ventricular pacing, with or without JTV519; SR were then isolated from dog left ventricular (LV) muscles. First, in JTV519-treated dogs, no signs of heart failure were observed after 4 weeks of chronic right ventricular pacing, LV systolic and diastolic functions were largely preserved, and LV remodeling was prevented. Second, JTV519 acutely inhibited both the FK506-induced Ca2+ leak from RyR in normal SR and the spontaneous Ca2+ leak in failing SR. Third, there was no abnormal Ca2+ leak in SR vesicles isolated from JTV519-treated hearts. Fourth, in JTV519-treated hearts, both the stoichiometry of FKBP12.6 binding to RyR and the amount of RyR-bound FKBP12.6 were restored toward the values seen in normal SR. Fifth, in JTV519-untreated hearts, RyR was PKA-hyperphosphorylated, whereas it was reversed in JTV519-treated hearts, returning the channel phosphorylation toward the levels seen in normal hearts. CONCLUSIONS: During the development of experimental heart failure, JTV519 prevented the amount of RyR-bound FKBP12.6 from decreasing. This in turn reduced the abnormal Ca2+ leak through the RyR, prevented LV remodeling, and led to less severe heart failure.


Assuntos
Insuficiência Cardíaca/prevenção & controle , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Tiazepinas/uso terapêutico , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Cães , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica , Transporte de Íons , Modelos Cardiovasculares , Miocárdio/metabolismo , Fosforilação , Retículo Sarcoplasmático/metabolismo , Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo/análise , Remodelação Ventricular
3.
Circulation ; 105(11): 1374-9, 2002 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-11901051

RESUMO

BACKGROUND: In heart failure, protein kinase A-mediated hyperphosphorylation of ryanodine receptors (RyRs) in sarcoplasmic reticulum (SR) causes dissociation of FKBP12.6 from RyRs. This results in an abnormal Ca2+ leak through RyRs, possibly leading to cardiac dysfunction. In the present study, we assess whether beta-blockers can correct this defect in RyR in tachycardia-induced heart failure and thereby improve cardiac function. METHODS AND RESULTS: SRs were isolated from dog left ventricular muscles (normal group, 4 weeks of rapid right ventricular pacing with or without propranolol [P(+) or P(-)]). End-diastolic and end-systolic diameters both increased less in P(+) than P(-), associated with a smaller decrease in fractional shortening in P(+). In SR from P(-), a prominent Ca2+ leak was observed, and FK506 (which dissociates FKBP12.6 from RyR) did not induce an additional Ca2+ leak. However, there was no appreciable Ca2+ leak in SR from P(+), although FK506 induced a Ca2+ leak as in normal SRs. In SR from P(+), an FK506-induced conformational change in RyR, which was virtually absent in SR from P(-), was observed as in normal SRs. Both the stoichiometry of FKBP12.6 versus RyR, assessed by [3H]FK506 and [3H]ryanodine binding assays, and the protein expression of FKBP12.6, assessed by Western blot analysis, were restored by propranolol toward the levels seen in normal SRs. CONCLUSIONS: Low-dose propranolol corrects the defective interaction of FKBP12.6 with RyR (restoration of RyR conformational change and prevention of Ca2+ leak from RyR), apparently resulting in an attenuation of intracellular Ca2+ overload and hence preventing the development of left ventricular remodeling in heart failure.


Assuntos
Insuficiência Cardíaca/prevenção & controle , Insuficiência Cardíaca/fisiopatologia , Miocárdio/metabolismo , Propranolol/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Cálcio/metabolismo , Estimulação Cardíaca Artificial , Diástole/efeitos dos fármacos , Modelos Animais de Doenças , Cães , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Hemodinâmica/efeitos dos fármacos , Técnicas In Vitro , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Sístole/efeitos dos fármacos , Tacrolimo/farmacologia , Remodelação Ventricular/efeitos dos fármacos
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