Activation of citrate synthase from a marine pseudomonad by adenosine monophosphate and potassium chloride

https://nsuworks.nova.edu/nsudigital_harrison/4028/thumbnail.jp

Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi

The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least-two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 A). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.Fil: Gonzalez, Soledad Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Valsecchi, Wanda Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Maugeri, Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Delfino, Jose Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Tetrameric and dimeric malate dehydrogenase isoenzymes in Trypanosoma cruzi epimastigotes

Two malate dehydrogenase isoforms, named MDH1 and MDH2, have been purified to homogeneity from Trypanosoma cruzi epimastigotes. Both enzymes consist of subunits with a molecular mass close to 33 kDa; native molecular mass determination by gel filtration, however, indicated that MDH1 is a dimer, whereas MDH2 is a tetramer. Both isoforms did not cross-react immunologically. The N-termini of both MDH isoforms and several tryptic peptides of MDH1 (amounting to about one third of the complete molecule) have been sequenced by automated Edman degradation. The tryptic digests of both enzymes have also been analysed by mass spectrometry (MALDI-TOF MS). The apparent K(m) values in both directions of the reaction have been determined, as well as the possible inhibition by excess of the substrate oxaloacetate. The sequence data, together with the pI values and the presence or absence of oxaloacetate inhibition indicate that the dimeric MDH1 is the mitochondrial isoenzyme, whereas the tetrameric MDH2 is the glycosomal isoenzyme. No evidence was found for the presence of a cytosolic isoform. (C) 2000 Elsevier Science B.V.Fil: Reynoso Hunter, Giselle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Hellman, Ulf. Ludwig Institute for Cancer Research; SueciaFil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Nowicki, Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Análise Preliminar de Cutículas Dispersas e Outros Microfragmentos Vegetais Encontrados em Carvões da Camada Morungava 6 e Folhelhos Carbonosos, Bacia Carbonífera de Gravataí, R.G.S. (Bacia do Paraná)

São analisados neste trabalho microfragmentos vegetais encontrados em associação com material palinológico resgatado em amostras de carvão da Camada Morungava 6 e folhelhos carbonosos (Bacia Carbonífera de Gravataí, Rio Grande do Sul), incluindo cutículas dispersas e restos de tecido xilemático, parenquimático e esclerenquimático. A análise quantitativa do material, feita de modo preliminar, revelou a predominância de cutículas relacionáveis a folhas e frutificações de Glossopteridales, Cordaitales e Sphenophyllales em comparação a fragmentos de tecidos vinculáveis a córtex e xilema. A classificação utilizada não teve por base um sistema taxonômico definido mas abrange, parcialmente, critérios sistemáticos adotados por MEYEN (1965), BOSE & MAHESHWARI (1968), CHANDRA & SURANGE (1975), NAVALE (1976) e MAITHY (1976)

An accurate record of volcanic ash fall deposition as characterized by dispersed organic matter in a lower Permian tonstein layer (Faxinal Coalfield, Paraná Basin, Brazil)

For the first time, the dispersed organic matter in the tonstein layer interbedded with a coal seam in the Faxinal Coalfield (Sakmarian, Southern Paraná Basin, Brazil) is characterized. The deposition of clusters of pollen grains was highly influenced by the intense ash fall process that probably occurred during seasonal dehiscence of reproductive structures. The well-preserved phytoclasts with their upper and lower leaf cuticles stuck together indicate that the rapid fall of ash on this material hindered organic biodegradation. The preservation of seemingly autochthonous Botryococcus colonies at the top of the tonstein layer is evidence of the subaqueous deposition of this layer. The darkening in cuticles and xylem phytoclasts can be attributed to different causes: the thermal influence of ash fall during deposition, chemical effects of the ash, prolonged oxidation of organic matter in low water level conditions or the burning of plant organs by wildfires. Analyses of dispersed organic matter along the tonstein layer showed that the organic matter succession reflects the composition of different plant strata (herbaceous pteridophytes and arboreal glossopterids-cordaitaleans) around the deposition site

An accurate record of volcanic ash fall deposition as characterized by dispersed organic matter in a Lower Permian tonstein bed (Faxinal Coalfield, Paraná Basin, Brazil)

For the first time, the dispersed organic matter in the tonstein layer interbedded with a coal seam in the Faxinal Coalfield (Sakmarian, Southern Paraná Basin, Brazil) is characterized. The deposition of clusters of pollen grains was highly influenced by the intense ash fall process that probably occurred during seasonal dehiscence of reproductive structures. The well-preserved phytoclasts with their upper and lower leaf cuticles stuck together indicate that the rapid fall of ash on this material hindered organic biodegradation. The preservation of seemingly autochthonous Botryococcus colonies at the top of the tonstein layer is evidence of the subaqueous deposition of this layer. The darkening in cuticles and xylem phytoclasts can be attributed to different causes: the thermal influence of ash fall during deposition, chemical effects of the ash, prolonged oxidation of organic matter in low water level conditions or the burning of plant organs by wildfires. Analyses of dispersed organic matter along the tonstein layer showed that the organic matter succession reflects the composition of different plant strata (herbaceous pteridophytes and arboreal glossopterids-cordaitaleans) around the deposition site

Screening and identification of metacaspase inhibitors: Evaluation of inhibition mechanism and trypanocidal activity

A common strategy to identify new antiparasitic agents is the targeting of proteases, due to their essential contributions to parasite growth and development. Metacaspases (MCAs) are cysteine proteases present in fungi, protozoa, and plants. These enzymes, which are associated with crucial cellular events in trypanosomes, are absent in the human host, thus arising as attractive drug targets. To find new MCA inhibitors with trypanocidal activity, we adapted a continuous fluorescence enzymatic assay to a medium-throughput format and carried out screening of different compound collections, followed by the construction of dose-response curves for the most promising hits. We used MCA5 from Trypanosoma brucei (TbMCA5) as a model for the identification of inhibitors from the GlaxoSmithKline HAT and CHAGAS chemical boxes. We also assessed a third collection of nine compounds from the Maybridge database that had been identified by virtual screening as potential inhibitors of the cysteine peptidase falcipain-2 (clan CA) from Plasmodium falciparum. Compound HTS01959 (from the Maybridge collection) was the most potent inhibitor, with a 50% inhibitory concentration (IC50) of 14.39mM; it also inhibited other MCAs from T. brucei and Trypanosoma cruzi (TbMCA2, 4.14mM; TbMCA3, 5.04mM; TcMCA5, 151mM). HTS01959 behaved as a reversible, slow-binding, and noncompetitive inhibitor of TbMCA2, with a mechanism of action that included redox components. Importantly, HTS01959 displayed trypanocidal activity against bloodstream forms of T. brucei and trypomastigote forms of T. cruzi, without cytotoxic effects on Vero cells. Thus, HTS01959 is a promising starting point to develop more specific and potent chemical structures to target MCAs.Fil: Perez, Brian Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bouvier, León Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agüero, Fernan Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Salas Sarduy, Emir. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Revisão fitoestratigrãfica do Grupo Itararé no Rio Grande do Sul: III - área de faxinal, Município de Guaíba, Rio Grande do Sul

The studied sequence outcrops at Faxinai locality, Barão do Triunfo sheet, and belongs to the silty-sand facies of the Itararé Group. The facies represents a lacustrine environment with periodical fluctuations of the borders. The taphoflorula consists of the genera Rubidgea, Paracalamites, Cordaites, Buriadia, Glossopteris, Cordaicarpus and Samaropsis. The palynological assemblage is composed of Punctatisporites, Granulatisporites, Calamospora, Leiotriletes, Cristatisporites (Pteridophyta), Potoniesporites and Pilacopollenites (Gymnospermae), the Glossopteridophyta scarcely being represented. Micro and macroflórula relationships are recognized, allowing the definitian of a Sakmarian age for the associations.A seqüência estudada corresponde a fácies areno-pelíticas do topo do Grupo Itararé na localidade de Faxinai, Folha Barão do Triunfo, RS e indica ambiente lacustre com periódicas expansões e recuos das bordas do lago. A tafoflórula é representada pelos gêneros Rubidgea, Paracalamites, Cordaites, Buriadia, Glossopteris, Gangamopteris, Cordaicarpus e Samaropsis. A associação palinológica é constituída principalmente por Puntatisporites, Granulatisporites, Calamospora, Leiotriletes, Cristatisporites (Pteridophyta), Potoniespohtes e Plicatipollenites (Gymnospermae), estando as Glossopteridophyta escassamente representadas. Sâo estabelecidas, neste trabalho, as vinculações entre a micro e macroflórula, indicando para as associações idade correspondente ao Sakmariano

A Tafoflora Triássica da Formação Santa Maria, RS, Brasil: III Dicroidium odontopteroides, Dicroidium zuberi e variações relacionadas a estas espécies

Em continuação ao estudo sistemático da tafoflora da Formação Santa Maria são descritas e identificas variações correspondentes às espécies Dicroidium odontopteróides (D. odontopteroides var. odontopteroides, D. odontopteroides var. moltenense e D. odontopteroides var. remotum) e Dicroidium zuberi (D. zuberi var. zuberi, D. suberi var. papillatum, D. zuberi var. feistmantelli e D. zuberi var. brasiliensis var. nov.). Estas taxas representam importantes elementos na caracterização bioestratigráfica da associação macroflorística estudada