A novel chlorpyrifos hydrolase CPD from Paracoccus sp. TRP: molecular cloning, characterization and catalytic mechanism

Background: Biodegradation is a reliable approach for efficiently eliminating persistent pollutants such as chlorpyrifos. Despite many bacteria or fungi isolated from contaminated environment and capable of degrading chlorpyrifos, limited enzymes responsible for its degradation have been identified, let alone the catalytic mechanism of the enzymes. Results: In present study, the gene cpd encoding a chlorpyrifos hydrolase was cloned by analysis of genomic sequence of Paracoccus sp. TRP. Phylogenetic analysis and BLAST indicated that CPD was a novel member of organophosphate hydrolases. The purified CPD enzyme, with conserved catalytic triad (Ser155-Asp251-His281) and motif Gly-Asp-Ser-Ala-Gly, was significantly inhibited by PMSF, a serine modifier. Molecular docking between CPD and chlorpyrifos showed that Ser155 was adjacent to chlorpyrifos, which indicated that Ser155 may be the active amino acid involved in chlorpyrifos degradation. This speculation was confirmed by site-directed mutagenesis of Ser155Ala accounting for the decreased activity of CPD towards chlorpyrifos. According to the key role of Ser155 in chlorpyrifos degradation and molecular docking conformation, the nucleophilic catalytic mechanism for chlorpyrifos degradation by CPD was proposed. Conclusion: The novel enzyme CPD was capable of hydrolyze chlorpyrifos and Ser155 played key role during degradation of chlorpyrifos.

Tyrosine phosphorylation of Kv1. 5 is upregulated in intrauterine growth retardation rats with exaggerated pulmonary hypertension

Intrauterine growth retardation (IUGR) is associated with the development of adult-onset diseases, including pulmonary hypertension. However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular dysfunction later in life is not fully understood. Here, we investigated the role of tyrosine phosphorylation of voltage-gated potassium channel 1.5 (Kv1.5) in this prenatal event that results in exaggerated adult vascular dysfunction. A rat model of chronic hypoxia (2 weeks of hypoxia at 12 weeks old) following IUGR was used to investigate the physiological and structural effect of intrauterine malnutrition on the pulmonary artery by evaluating pulmonary artery systolic pressure and vascular diameter in male rats. Kv1.5 expression and tyrosine phosphorylation in pulmonary artery smooth muscle cells (PASMCs) were determined. We found that IUGR increased mean pulmonary artery pressure and resulted in thicker pulmonary artery smooth muscle layer in 14-week-old rats after 2 weeks of hypoxia, while no difference was observed in normoxia groups. In the PASMCs of IUGR-hypoxia rats, Kv1.5 mRNA and protein expression decreased while that of tyrosine-phosphorylated Kv1.5 significantly increased. These results demonstrate that IUGR leads to exaggerated chronic hypoxia pulmonary arterial hypertension (CH-PAH) in association with decreased Kv1.5 expression in PASMCs. This phenomenon may be mediated by increased tyrosine phosphorylation of Kv1.5 in PASMCs and it provides new insight into the prevention and treatment of IUGR-related CH-PAH.

Acute effects of soft drink intake on calcium and phosphate metabolism in immature and adult rats

Objetivo. Probar los efectos de la ingestión aguda de un refresco de cola que contiene ácido fosfórico sobre el metabolismo ácido-base y de calcio y fosfatos. Material y métodos: Se estudiaron 14 ratas adultas de 90 días de edad de la cepa Sprague Dawley y 14 inmaduras de 30 días de edad. Aleatoriamente se asignaron siete animales de cada grupo para recibir sólo agua (grupos control) o Coca-Cola ad libitum durante siete días. Después los animales se colocaron individualmente en jaulas metabólicas para colectar orina de 24 hrs. Los animales se exsanguinaron por punción directa de la arteria aorta y se midió pH y calcio en sangre total. Se midieron niveles plasmáticos de PTH, 1Ó, 25 (OH)2 D3 y 25 OH d3 con estuches comerciales de IRMA y RIA y plasmáticos y urinarios de creatinina, fosfatos y calcio. Resultados. En ambos grupos, los animales que recibieron refresco desarrollaron hipercalciuria e hiperfosfaturia. En los animales inmaduros el pH disminuyó de 7.45 ñ 0.04 a 7.33 ñ 0.02 (p< 0.05) pero co cambió en los animales adultos. En los animales inmaduros el calcio ionizado disminuyó significativamente de 1.06 ñ 0.04 a 0.80 ñ 0.06 meq/l(p < 0.05), pero no en los adultos, Sólo los animales adultos desarrollaron hiperparatiroidismo significativo. Los animales inmaduros mostraron trastornos más graves del metabolismo del calcio y fosfato relacionados con la ingestion de refrescos de cola

An extracellular phosphatase from Aspergillus fumigatus.

A potential producer of extracellular phosphatase has been isolated and identified as A. fumigatus. The fungal phosphatase is active in pH range 5 to 8 and its temperature optimum is 65 degrees C. The mineralisation of organic phosphates present in Neem cake and press mud by this enzyme has been demonstrated.

Difference in the sensitivity of junctional and longitudinal sarcoplasmic reticulum Ca(2+)-ATPase to ADP

The Ca2+ release mechanism that triggers muscle contraction is still not completely understood. We compared Ca2+ accumulation and acetyl phosphate hydrolysis by the Ca(2+)-ATPases present in the longitudinal and junctional membrane of the sarcoplasmic reticulum of rabbit skeletal muscle and found that Ca(2+)-ATPase is more sensitive to ADP inhibition when the enzyme is located on the junctional membrane than when the enzyme is located on the longitudinal membrane (K0.5 = 144 microM for the junctional enzyme vs K0.5 = 415 microM for the longitudinal enzyme). When the enzyme was solubilized in non-ionic detergent (2% v/v Triton X-100) and tested again using 2 mM AcP as substrate, the difference in ADP sensitivity observed with native preparations disappeared. We conclude that the enzyme is regulated differently depending on its localization on the membrane of the sarcoplasmic reticulum