RESUMO
Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.
Assuntos
Drosophila melanogaster/enzimologia , Peptídeo Hidrolases/química , Complexo de Endopeptidases do Proteassoma , Ubiquitinas/metabolismo , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero/enzimologia , Substâncias Macromoleculares , Microscopia Eletrônica de Transmissão e Varredura , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/ultraestrutura , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Ubiquitina TiolesteraseRESUMO
Preparations of rat detrusor vesicae urinariae exposed to 50 kV X-irradiation with 10 to 200 Gy (single dose) at dose-rates of 30 and 60 Gy/min reacted immediately with a dose and dose-rate dependent reversible or persistent increase (up to ten hours) of the basal tone and an increase or a decrease of the acetylcholine contractile response. The motor activity was recorded isotonically. For measurements of time changes following treatment in vivo the bladder was locally irradiated from lateral position with single 300 kV X-ray doses of 10, 25 and 50 Gy. The motor reaction of isolated detrusor preparations to acetylcholine had a threshold concentration in control animals of 2.3 X 10(-10) mol/l (n = 33); the sensitivity to acetylcholine was diminished as early as one to two hours after local irradiation with 50 Gy as reflected in a ten times higher threshold concentration, which decreased further with time past treatment up to 40 days. The inhibitory effect after 25 Gy was weaker. The contractile response of acetylcholine at different concentrations (10(-10) to 10(-5) mol/l) was also diminished after irradiation (50 Gy). It is suggested that the pathophysiological reactions of the radiogenic bladder are based on multifactorial mechanisms and X-ray induced tonic contraction as well as inhibition of the acetylcholine contractile response could be essential factors for the clinically observed hypertonia of the irradiated bladder ("radiogene Harnblase") and its functional volume decrease as well as of the diminished pressure during micturition.
Assuntos
Bexiga Urinária/efeitos da radiação , Acetilcolina/farmacologia , Animais , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/efeitos da radiação , Perfusão/métodos , Ratos , Ratos Endogâmicos , Fatores de Tempo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiopatologiaRESUMO
Etiology, pathophysiology, and clinical picture of local frost-injuries are described. By aid of literature, and by proper experiences in 49 patients, who had been treated at the University-Hospital of Innsbruck during the year of 1966 to 1973, therapy of local frost-injuries is discussed. Moreover the advantage of surgical therapy on the sympathicus over conservative treatment is pointed out.
