The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer.

Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.

Reaction of wild-type C365S, and C458S saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with fluorescent iodoacetamide derivatives.

The reactivities of Cys365 and Cys458 of ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase against a range of sulfhydryl reagents have been investigated. The effect of pH on the second order reaction constants of N-(1-pyrenyl)maleimide with mutant C458S and C365S PEP carboxykinases allowed the determination of pKa values of 9.4 and 9.1 for Cys365 and Cys458, respectively. The analysis of the inactivation rates of C458S and C365S mutant enzymes by several sulfhydryl reagents of different hydrophobicity showed that the microenvironment of these residues is rather polar. Anisotrophy measurements and acrylamide quenching experiments carried out with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled mutant enzymes indicated a higher rotational freedom and solvent exposure for the probe linked to Cys458 than to Cys365. These findings point to differences in the protein microenvironments around Cys365 and Cys458 in S. cerevisiae PEP carboxykinase. A comparison of the results obtained with published data for GTP-dependent PEP carboxykinases, suggest significant differences for the protein region around the reactive cysteinyl residues in these enzymes.

Bacteriocin Typing of Burkholderia (Pseudomonas) solanacearum Race 1 of the French West Indies and Correlation with Genomic Variation of the Pathogen.

Burkholderia solanacearum race 1 isolates indigenous to the French West Indies were characterized by bacteriocin typing and two genomic fingerprinting methods: pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE) and PCR with primers corresponding to repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX elements (collectively known as rep-PCR). The survey comprised 24 reference strains and 65 isolates obtained from a field trial in Guadeloupe in 1993. Comparison of the data identified RC-PFGE as the most discriminatory method, delineating 17 pulsed-field gel profile types. rep-PCR and bacteriocin typing identified nine rep-PCR profile types and nine bacteriocin groups. Independent determination of similarity coefficients and clustering of RC-PFGE and rep-PCR data identified six groups common to both sets of data that correlated to biovar and bacteriocin groups. Further study of bacteriocin production in planta gave results consistent with in vitro bacteriocin typing. It was observed that spontaneous bacteriocin-resistant mutants exhibited a cross-resistance to other bacteriocins as identified by the typing scheme and that such mutants possessed a selective advantage for growth over isogenic nonmutants in the presence of a bacteriocin. The results are significant in the search for biological control of disease by nonpathogenic mutants of the wild-type organism.

Visual discrimination and visuomotor integration among two classes of Brazilian children.

The Raven Matrices, Beery, and Bender-Gestalt tests were given to 415 Brazilian children in public and private schools corresponding to two socioeconomic statuses. Both social class and grade in school influenced all scores more than age. The Raven Matrices appeared to be the most affected by social class differences. Visual-motor integration and visual discrimination skills were moderately (.47-.69) correlated, especially among the private school students. Maturational differences based on age and school grade were considered somewhat better assessed by the Beery than the Raven or Bender-Gestalt tests. The wide age range at each grade and the decreasing enrollment in higher grades makes interpretation difficult.

Autismo, psicanálise e desordem cerebral: uma crítica / Autism psychoanalysis and cerebral disorder: a critique

Com base em recentes descobertas científicas referentes à etiologia do autismo, o artigo discute e avalia os métodos da ciência empírica em confronto com os da psicanálise. As origens e tradiçöes diversas destas duas orientaçöes säo contrapostas e säo discutidas as implicaçöes das mesmas sobre a análise e o tratamento da doença mental. Assinala a falha geral do enfoque psicanalítico em satisfazer os requisitos dos métodos da ciência empírica. Säo apontados também críticas específicas, inclusive dos próprios psicanalistas

Autismo, psicanalise e desordem cerebral: uma critica

Com base em recentes descobertas cientificas refentes a etiologia do autismo, o artigo discute e avalia os metodos da ciencia empirica em confronto com os da psicanalise. As origens e tradicoes diversas destas duas orientacoes das mesmas sobre a analise e o tratamento da doenca mental. Assinala a falha geral do enfoque psicanalitico em satisfazer os requisitos dos metodos da ciencia empirica. Sao apontadas tambem criticas especificas, inclusive dos proprios psicanalistas.