Capillary electrophoresis and mutational images of hemoglobin sendagi [Β42 (CD1) PHE → VAL; HBB: C.127T→G].

We report two cases of hemoglobin Sendagi in a Romanian family residing in Spain: a four-year-old boy and his mother, who had been previously diagnosed with another type of congenital hemolytic anemia and had undergone splenectomy in her country during childhood. The unstable hemoglobin variant, hemoglobin Sendagi, is characterized by decreased oxygen affinity caused by replacement of one of the critical amino acid residues, phenylalanine beta 42 (CD1) of the beta-chain, with valine in the heme pocket, resulting in methemoglobin formation. As a result of migratory movements in Europe, new disease-causing hemoglobin variants are emerging in our country. Here, capillary electrophoresis enabled the identification of the variant and a molecular study was used to establish an accurate diagnosis.

A tricistronic human adrenodoxin reductase-adrenodoxin-cytochrome P450 27A1 vector system for substrate hydroxylation in Escherichia coli.

Cytochrome P450 (P450) 27A1 catalyzes 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D(3), serving as an important component for the maintenance of lipid homeostasis. In eukaryotic cells P450 27A1 is a membrane-bound protein located on the inner mitochondrial membrane and requires two auxiliary reduction partners, adrenodoxin (Adx) and NADPH-adrenodoxin reductase (Adr), for catalysis in the bile acid biosynthesis pathway. A strategy was developed for the functional coexpression of P450 27A1 with Adr and Adx in a tricistronic fashion (single RNA, three proteins) in Escherichia coli, mimicking the mitochondrial P450 system. Intact bacterial cells coexpressing the P450 vector (pTC27A1) efficiently hydroxylated cholesterol at the 27 position as well as vitamin D(3) at the 25 position when supplemented with glycerol as a carbon source. Thus, E. coli containing pTC27A1 is able to hydroxylate cholesterol in a self-sufficient fashion and is suitable for further applications of protein interaction, drug discovery, and inhibitor evaluation and for the study of other mitochondrial P450s and oxysterol production in microorganisms without a need for membrane reconstitution, membrane simulation by detergents, or purification of the components.

Approaches to deorphanization of human and microbial cytochrome P450 enzymes.

One of the general problems in biology today is that we are characterizing genomic sequences much faster than identifying the functions of the gene products, and the same problem exists with cytochromes P450 (P450). One fourth of the human P450s are not well-characterized and therefore considered "orphans." A number of approaches to deorphanization are discussed generally. Several liquid chromatography-mass spectrometry approaches have been applied to some of the human and Streptomyces coelicolor P450s. One current limitation is that too many fatty acid oxidations have been identified and we are probably missing more relevant substrates, possibly due to limits of sensitivity.

Characterizing proteins of unknown function: orphan cytochrome p450 enzymes as a paradigm.

With the rapid completion of genomic sequences of organisms today, we have far more gene products than functions we can ascribe. A number of experimental strategies have been developed and applied, both in vitro and in vivo, to put functions to these orphan proteins. The "deorphanization" of human and Streptomyces cytochrome P450 enzymes is considered quite important for pharmacology, with ramifications for the use of clinical therapeutics. The myriad of possibilities is too enormous to screen one reaction at a time, thus metabolomic or proteomic screens with complex biological samples are promising current strategies.

Correlation of the genotoxic activation and kinetic properties of Salmonella enterica serovar Typhimurium nitroreductases SnrA and cnr with the redox potentials of nitroaromatic compounds and quinones.

Bacterial nitroreductases (NRs) catalyse the oxygen-insensitive reduction of several nitro-substituted compounds and quinones. SnrA and cnr NRs have been previously identified in Salmonella enterica serovar Typhimurium; they reduce several environmental nitro compounds that display mutagenic activity in the Ames test. Although some of their biochemical properties have been reported, the substrate specificity of each protein over mutagenic nitro compounds is unknown; even more, the possible relationship between their capacity to activate nitro compounds into mutagens and the redox properties of putative substrates has been poorly investigated. We have purified SnrA and cnr and investigated their capacity to activate several mutagens in the Ames test as well as their kinetic parameters K(m) and V(max). Our results show that SnrA and cnr are able to activate 2,7-dinitrofluorene with the same efficiency and a similar mutagenic potency in the YG7132 tester strain; 1-nitropyrene and 1,3-dinitropyrene were efficiently activated by cnr, whereas 1,8-dinitropyrene, 1,6-dinitropyrene and 2-nitrofluorene were scarcely activated by either NR. The mutagenic potency of nitro compounds obtained in the presence of either enzyme correlates with their redox potential reported in the literature. On the other hand, a good correlation was obtained between the catalytic efficiency (V(max)/K(m)) of the purified cnr with the redox potential of eight molecules including nitro-substituted compounds and quinones. No correlation between redox potential and catalytic efficiency by SnrA was observed, suggesting that factors other than redox potential such as the structure of the compounds are involved in the catalytic efficiency of SnrA.

[Spontaneous bacterial peritonitis caused by Listeria in a patient with cirrhosis: Case report]. / Peritonitis bacteriana espontánea por Listeria monocytogenes, en un paciente con cirrosis hepática: Caso clínico.

Infections caused by Listeria monocytogenes are usually found in cattle and occasionally appear in humans, particularly pregnant women and immunocompromised individuals. Peritonitis by Listeria monocytogenes is a rare but dangerous condition that must be recognized early, since it requires a specific treatment. We report a 31 year-old male with alcoholic cirrhosis that developed ascites with abdominal pain and fever. The peritoneal fluid culture yielded Listeria monocytogenes. The patients was initially treated with cefotaxim and later with ampicillin and levofloxacin. The patient voluntarily abandoned treatment and died at home two weeks later.

Nitrocompound activation by cell-free extracts of nitroreductase-proficient Salmonella typhimurium strains.

A characterization of nitrocompounds activation by cell-free extracts (CFE) of wild-type (AB(+)), SnrA deficient (B(+)), Cnr deficient (A(+)) and SnrA/Cnr deficient (AB(-)) Salmonella typhimurium strains has been done. The Ames mutagenicity test (S. typhimurium his(+) reversion assay) was used, as well as nitroreductase (NR) activity determinations where the decrease in absorbance generated by nitrofurantoin (NFN) reduction and NADP(H) oxidation in the presence of NFN, nitrofurazone (NFZ), metronidazole (MTZ) and 4-nitroquinoline-1-oxide (4NQO) were followed. Different aromatic and heterocyclic compounds were tested for mutagenic activation: 2-nitrofluorene (2-NF); 2,7-dinitrofluorene (2,7-DNF); 1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP); 1,6-dinitropyrene (1,6-DNP); and 1,8-dinitropyrene (1,8-DNP). Differential mutagenicity was found with individual cell free extracts, being higher when the wild type or Cnr containing extract was used; nevertheless, depending on the nitrocompound, activation was found when either NR, SnrA or Cnr, were present. In addition, all nitrocompounds were more mutagenic after metabolic activation by CFE of NR proficient strains, although AB(-) extract still showed activation capacity. On the other hand, NR activity was predominantly catalyzed by wild type CFE followed by A(+), B(+) and AB(-) extracts in that order. We can conclude that results from the Ames test indicate that Cnr is the major NR, while NFN and NFZ reductions were predominantly catalyzed by SnrA. The characterization of the residual NR activity detected by the mutagenicity assay and the biochemical determinations in the AB(-) CFE needs further investigation.

Peritonitis bacteriana espontánea por Listeria monocytogenes, en un paciente con cirrosis hepática: Caso clínico / Spontaneous bacterial peritonitis caused by Listeria in a patient with cirrhosis: Case report

Infections caused by Listeria monocytogenes are usually found in cattle and occasionally appear in humans, particularly pregnant women and immunocompromised individuals. Peritonitis by Listeria monocytogenes is a rare but dangerous condition that must be recognized early, since it requires a specific treatment. We report a 31 year-old male with alcoholic cirrhosis that developed ascites with abdominal pain and fever. The peritoneal fluid culture yielded Listeria monocytogenes. The patients was initially treated with cefotaxim and later with ampicillin and levofloxacin. The patient voluntarily abandoned treatment and died at home two weeks later.

Niveles de progesterona y dinámica folicular en el posparto de vacas Holstein en condiciones del trópico bajo Colombiano / Progesterone levels and follicular dynamic during Holstein cows postpartum under low tropic conditions in Colombia

Trece vacas Holstein seleccionadas al azar se siguieron desde el día 7 posparto, cada tres días, hasta su preñez o día 120 posparto, determinando la reactivación ovárica mediante la caracterización de dinámica folicular (ultrasonografía) y los niveles de progesterona, mediante Radioinmunoanálisis (RIA). Los intervalos parto-primer estro observado, primera inseminación, primera ovulación, primer aumento de progesterona (>1 ng/ml) y primer folículo dominante fueron 67.3, 70.3, 27, 36 y 26 días respectivamente; la tasa de concepción a primera inseminación fue 54,4%. Los patrones de crecimiento folicular en los primeros 15 días posparto fueron inconsistentes, presentando algunas hembras, en forma indeterminada, ovarios inactivos y otras folículos con diámetros mayores de 10 mm. El 72,6% de las vacas presentaron celos silentes, con fases luteales más cortas que aquellas con celo manifiesto (12 vs. 15 días) y concentración de progesterona al día 15 así mismo inferior (1.994 vs. 3.3873 ng/ml). En vacas preñadas se observó un inicio de la actividad luteal más temprano (día 3) que en vacas vacías (día 6), teniendo las primeras el día 15 niveles de progesterona mayores a 3 ng/ml, mientras en las otras descendía paulatinamente, demostrando luteolisis temprana. Los resultados permiten determinar que el inicio de la reactivación ovárica (dinámica folicular y hormonal) no está determinada por la presentación del primer estro manifiesto, siendo el uso del RIA un apoyo para detectar la ciclicidad en ausencia de signos externos de estro. Para la explotación en estudio, el retraso en el comienzo de la actividad reproductiva, evaluado por el amplio rango de días abiertos y el número de servicios por preñez, no es debido a la falta de una dinámica ovárica temprana, siendo necesario analizar conjuntamente otros factores en futuras investigaciones, como la presión productiva, problemas nutricionales u otros, que puedan estar afectando la eficiencia reproductiva...

Evaluación de la actividad scavenger frente a los radicales superóxido e hidroxilo por parte de algunos compuestos obtenidos de plantas medicinales colombianas / Scavanger activity to superoxide, peroxida and hydroxil radicals of several compounds obtained from Colombian medicinal plants

Se realizó un estudio para evaluar la posible actividad scavenger de 6 compuestos provenientes de plantas medicinales colombianas frente a los radicales superóxido, peroxilo e hidroxilo, mediante el uso de técnicas generadoras y atrapadoras de especies de oxígeno reactivas. Se comprobó la actividad antirradicalaria de la umbeliferona y la hesperidina, la mejor captación de radical peroxilo por khellina, escopolina y umbeliferona. La arbutina y el pinitol mostraron los mejores resultados frente al radical hidroxilo. Cinco de los seis compuestos mostraron resultados promisorios frente al radical superóxido

Alcaloides isoquinolínicos de la corteza y flores de Erythrina fusca Loureiro / Isoquinolinic alcaloids from the bark and flowers of the Erythrina fusca Loureiro

De la corteza y flores de Erythrina fusca Loureiro se aislaron los alcaloides isoquinolínicos (+) -epieritratidina y 8-(+)-oxoerisodina, los cuales fueron identificados con base en sus constantes espectroscópicas. Se determinó la actividad antimicrobiana de extractos crudos y fracciones frente a bacterias y hongos. Se evaluó la actividad farmacólogica de extractos crudos y fracciones frente a ratas tipo WISTAR

Comprobación de la actividad hipoglicemiante y captadora de radicales libres oxigenados de los principios activos de Curatella americana L / Comprobation of the hypoglycemic and capture of oxigenated of radical free of the active principles of Curatella american L

La actividad hipoglicemiante - antidiabética de una fracción del extracto clorofórmico de la corteza de Curatella americana L. ("chaparro") fue evaluada en ratones normoglicémicos y en ratas diabéticas por aloxano, en ensayos agudos y de administración crónica. Junto con los niveles de glicemia se determinó el nivel de insulina (Enzymun - Test insulina, técnica ELISA). Se probó también la actividad contra radical superóxido (Sistema Xantina / Xantina Oxidasa), radical hidroxilo (sistema H2O2/Fe a la 3 - EDTA/Ascorbato) y ácido hipocloroso (Sistema NaOCl / H2SO4). Se encontró un importante efecto antihiperglicemiante, un efecto protector contra la diabetes aloxánica y disminución de la glicemia en el estado diabético. Los extractos mostraron una marcada actividad contra radical superóxido y menos importante contra HOCl. La actividad contra radicales hidroxilo y peroxilo estuvo interferida por el vehículo (MeOH). La actividad antidiabética del extracto podría estar mediada por los supuestos atrapadores de "Especies Oxígeno -Reactivas"