RESUMO
In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15-70 degrees C). Specific activities were determined toward choline chloride (4.70 +/- 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 +/- 0.45 x 10(-2) U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 +/- 0.12 x 10(-2) U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K (M) = 1.51 +/- 0.09 mM and V (max) = 42.73 +/- 0.42 mU/min for choline chloride and K (M) = 4.77 +/- 0.76 mM and V (max) = 48.40 +/- 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Arthrobacter/enzimologia , Oxirredutases do Álcool/química , Arthrobacter/genética , Arthrobacter/isolamento & purificação , Betaína/análogos & derivados , Betaína/metabolismo , Colina/metabolismo , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de DNA , Microbiologia do Solo , Especificidade por Substrato , TemperaturaRESUMO
Mental health care shortages in rural areas have resulted in the majority of services being offered through primary medical care settings. The authors argue that a paradigm shift must occur so that those in need of mental health care have reasonable, timely access to these services. Changes proposed include integrating mental health services into primary medical care settings, moving away from the traditional view of mental health care services (one therapist, one hour, and one client), and increasing the consultative role of psychologists and other mental health care providers in primary medical care. Characteristics of mental health providers that facilitate effective integration into primary medical care are presented. The results of a needs assessment survey and an example of a telemental health project are described. This project involved brief consultations with patients and their physicians from a shared care model using a broadband internet telecommunications link between a rural clinic and mental health service providers in an urban area.