Modelagem molecular comparativa da enzima Adenosina Kinase isoforma 1 (AK1) de Schistosoma mansoni

O Schistosoma mansoni, parasito responsável pela esquistossomose mansônica, depende integralmente da via de salvação de purinas para o suprimento das bases púricas. Uma vez que o parasito não possui a via se sí­ntese dessas bases, a via de salvação representa um alvo potencial para o desenvolvimento de novos fármacos. A adenosina kinase (AK) é uma das principais enzimas dessa via na sí­ntese direta de adesinosina monofosfato (AMP) a partir de adenosina. No genoma do parasito foram identificadas duas isoformas da AK, no entanto, apenas a estrutura tridimensional de SmAK2 (Smp_008360) foi resolvida experimentalmente. Assim, a modelagem comparativa foi utilizada como ferramenta para predizer a estrutura da isoforma SmAK1 (Smp_008400) e possibilitar a análise comparativa entre as duas estruturas. A modelagem foi realizada através do software Swiss-Model, o refinamento no 3Drefine, a validação no PDBsum, a observação e análise da estrutura e a comparação entre as isoformas no PyMOL. As estruturas tridimensionais da SmAK1 e SmAK2 possuem 80% de identidade e as sobreposições obtiveram RMSDs de 0.155 Å e de 0.167 Å atestando a similaridade do enovelamento entre ambas. Os modelos gerados possuem 343 resí­duos de aminoácidos e os gráficos de Ramachandran demonstram que 94,1% dos resí­duos estão em regiões favoráveis, 5,5% em regiões adicionalmente permitidas e 0,3% em regiões não permitidas garantindo a confiabilidade dos modelos gerados. As análises comparativas do sí­tio ativo da AK humana com relação a SmAK1, demonstram pouca variação nos aminoácidos que os constituem, como a substituição da Ser65 na AK humana pelo resí­duo equivalente Ala79 na SmAK1 e a não interação da adenosina com a Ala136 na humana, que corresponderia aos resí­duos funcionais Thr150 da SmAK1 e Thr136 da SmAK2 dos sí­tios ativos. Os resultados obtidos sugerem que dados de ensaios cinéticos com ligantes e possí­veis inibidores sejam realizados para complementar os dados obtidos e embasar conclusões sobre a viabilidade da enzima como alvo potencial para o desenvolvimento de novos fármacos

Modelagem molecular comparativa da enzima Adenosina Kinase isoforma 1 (AK1) de Schistosoma mansoni

O Schistosoma mansoni, parasito responsável pela esquistossomose mansônica, depende integralmente da via de salvação de purinas para o suprimento das bases púricas. Uma vez que o parasito não possui a via se síntese dessas bases, a via de salvação representa um alvo potencial para o desenvolvimento de novos fármacos. A adenosina kinase (AK) é uma das principais enzimas dessa via na síntese direta de adesinosina monofosfato (AMP) a partir de adenosina. No genoma do parasito foram identificadas duas isoformas da AK, no entanto, apenas a estrutura tridimensional de SmAK2 (Smp_008360) foi resolvida experimentalmente. Assim, a modelagem comparativa foi utilizada como ferramenta para predizer a estrutura da isoforma SmAK1 (Smp_008400) e possibilitar a análise comparativa entre as duas estruturas. A modelagem foi realizada através do software Swiss-Model, o refinamento no 3Drefine, a validação no PDBsum, a observação e análise da estrutura e a comparação entre as isoformas no PyMOL. As estruturas tridimensionais da SmAK1 e SmAK2 possuem 80% de identidade e as sobreposições obtiveram RMSDs de 0.155 Å e de 0.167 Å atestando a similaridade do enovelamento entre ambas. Os modelos gerados possuem 343 resíduos de aminoácidos e os gráficos de Ramachandran demonstram que 94,1% dos resíduos estão em regiões favoráveis, 5,5% em regiões adicionalmente permitidas e 0,3% em regiões não permitidas garantindo a confiabilidade dos modelos gerados. As análises comparativas do sítio ativo da AK humana com relação a SmAK1, demonstram pouca variação nos aminoácidos que os constituem, como a substituição da Ser65 na AK humana pelo resíduo equivalente Ala79 na SmAK1 e a não interação da adenosina com a Ala136 na humana, que corresponderia aos resíduos funcionais Thr150 da SmAK1 e Thr136 da SmAK2 dos sítios ativos. Os resultados obtidos sugerem que dados de ensaios cinéticos com ligantes e possíveis inibidores sejam realizados para complementar os dados obtidos e embasar conclusões sobre a viabilidade da enzima como alvo potencial para o desenvolvimento de novos fármacos.

Age-related renal parenchymal lesions in children with first febrile urinary tract infections

The aim of this study was to define the association between age and the occurrence of acute pyelonephritis and renal scars

Association of procalcitonin with acute pyelonephritis and renal scars in pediatric UTI.

Procalcitonin was a more robust predictor compared with C-reactive protein or white blood cell count for selectively identifying children who had APN during the early stages of UTI, as well as those with late scarring

Procalcitonin to reduce the number of unnecessary cystographies in children with a urinary tract infection: A European Validation Study

Objective To validate high serum procalcitonin (PCT) as a predictor of vesicoureteral reflux (VUR) in children with a first febrile urinary tract infection (UTI). Study design This secondary analysis of prospective hospital-based cohort studies included children ages 1 month to 4 years with a first febrile UTI. Results Of the 398 patients included in 8 centers in 7 European countries, 25% had VUR. The median PCT concentration,vas significantly higher in children with VUR than in those without: 1.6 versus 0.7 ng/ml, (P = 10(-4)). High PCT (>= 0.5 ng/ml) was' associated with VUR (OR: 2.3. 95% Cl, 1.3 to 3.9; P = 10(-3)). After adjustment for all cofactors, the association remained significant (OR: 2.5: 95% Cl, 1.4 to 4.4; P = 10(-3)). The strength of the relation increased with tire grade of reflux (P = 10(-5).). The sensitivity of procalcitonin was 75% (95% Cl, 66 to 83) for all-grade VUR and 100% (95% Cl, 81 to 100) for grade >= 4 VUR. both with 43% specificity (95% Cl, 37 to 48). Conclusions High PCT is a strong, independent and now validated predictor of VUR that can be used to identify low-risk patients and thus avoid one third of the unnecessary cystourethrographies in children with a first febrile UTI

Global analysis of erythroid cells redox status reveals the involvement of Prdx1 and Prdx2 in the severity of beta thalassemia.

β-thalassemia is a worldwide distributed monogenic red cell disorder, characterized by an absent or reduced beta globin chain synthesis. The unbalance of alpha-gamma chain and the presence of pathological free iron promote severe oxidative damage, playing crucial a role in erythrocyte hemolysis, exacerbating ineffective erythropoiesis and decreasing the lifespan of red blood cells (RBC). Catalase, glutathione peroxidase and peroxiredoxins act together to protect RBCs from hydrogen peroxide insult. Among them, peroxiredoxins stand out for their overall abundance and reactivity. In RBCs, Prdx2 is the third most abundant protein, although Prdxs 1 and 6 isoforms are also found in lower amounts. Despite the importance of these enzymes, Prdx1 and Prdx2 may have their peroxidase activity inactivated by hyperoxidation at high hydroperoxide concentrations, which also promotes the molecular chaperone activity of these proteins. Some studies have demonstrated the importance of Prdx1 and Prdx2 for the development and maintenance of erythrocytes in hemolytic anemia. Now, we performed a global analysis comparatively evaluating the expression profile of several antioxidant enzymes and their physiological reducing agents in patients with beta thalassemia intermedia (BTI) and healthy individuals. Furthermore, increased levels of ROS were observed not only in RBC, but also in neutrophils and mononuclear cells of BTI patients. The level of transcripts and the protein content of Prx1 were increased in reticulocyte and RBCs of BTI patients and the protein content was also found to be higher when compared to beta thalassemia major (BTM), suggesting that this peroxidase could cooperate with Prx2 in the removal of H2O2. Furthermore, Prdx2 production is highly increased in RBCs of BTM patients that present high amounts of hyperoxidized species. A significant increase in the content of Trx1, Srx1 and Sod1 in RBCs of BTI patients suggested protective roles for these enzymes in BTI patients. Finally, the upregulation of Nrf2 and Keap1 transcription factors found in BTI patients may be involved in the regulation of the antioxidant enzymes analyzed in this work

Procalcitonin is a predictor for high-grade vesicoureteral reflux in children: Meta-analysis of individual patient data

Objective: To assess the predictive value of procalcitonin, a serum inflammatory marker, in the identification of children with first urinary tract infection (UTI) who might have high-grade (≥3) vesicoureteral reflux (VUR). Study design: We conducted a meta-analysis of individual data, including all series of children aged 1 month to 4 years with a first UTI, a procalcitonin (PCT) level measurement, cystograms, and an early dimercaptosuccinic acid scan. Results: Of the 152 relevant identified articles, 12 studies representing 526 patients (10% with VUR ≥3) were included. PCT level was associated with VUR ≥3 as a continuous (P =.001), and as a binary variable, with a 0.5 ng/mL preferred threshold (adjusted OR, 2.5; 95% CI, 1.1 to 5.4). The sensitivity of PCT ≥0.5 ng/mL was 83% (95% CI, 71 to 91) with 43% specificity rate (95% CI, 38 to 47). In the subgroup of children with a positive results on dimercaptosuccinic acid scan, PCT ≥0.5 ng/mL was also associated with high-grade VUR (adjusted OR, 4.8; 95% CI, 1.3 to 17.6). Conclusions: We confirmed that PCT is a sensitive and validated predictor strongly associated with VUR ≥3, regardless of the presence of early renal parenchymal involvement in children with a first UTI. © 2011 Mosby Inc. All rights reserved

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKCδ) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKCδ translocates to the nucleus, PKCδ and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKCδ and Runx2 function