RESUMO
Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5'-->3' direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 degrees C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 degrees C) and PPN (35-40 degrees C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of beta-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification.
Assuntos
Aminoidrolases/genética , Proteínas de Arabidopsis , Arabidopsis/genética , Hidroliases/genética , Aminoidrolases/metabolismo , Arabidopsis/metabolismo , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Germinação , Glucuronidase , Hidroliases/metabolismo , Concentração de Íons de Hidrogênio , Ácidos Indolacéticos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Família Multigênica , Mutação , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Especificidade por Substrato , TemperaturaAssuntos
Contratura , Traumatismos dos Dedos/terapia , Articulações dos Dedos , Contenções , HumanosRESUMO
The Worker Role Interview is a semistructured interview designed to be used as the psychosocial-environmental component of the initial rehabilitation assessment for the injured worker. Interrater reliability of the Worker Role Interview was computed for three raters for a sample of 30 adult subjects receiving rehabilitation due to an upper extremity injury. Reliability was assessed with the intraclass correlation approach. The coefficients estimating interrater reliability for six content areas ranged from .46 to .92 with a total value of .81. Three out of six content areas received ratings well below the accepted standard of .80, suggesting further need for instrument refinement in select areas. Test-retest reliability was computed for one rater for a sample of 20 subjects. The intraclass coefficient values ranged from .86 to .94 with a total value of .95 indicating high test-retest reliability.