Peripheral Blood Leukocytes And Serum Nested Polymerase Chain Reaction Are Complementary Methods For Monitoring Active Cytomegalovirus Infection In Transplant Patients.

Human cytomegalovirus is an important cause of morbidity and mortality in immunocompromised patients. Qualitative polymerase chain reaction (PCR) has proven to be a sensitive and effective technique in defining active cytomegalovirus infection, in addition to having low cost and being a useful test for situations in which there is no need for quantification. Real-time PCR has the advantage of quantification; however, the high cost of this methodology makes it impractical for routine use. To apply a nested PCR assay to serum (sPCR) and to evaluate its efficiency to diagnose active cytomegalovirus infection compared with PCR of peripheral blood leukocytes (L-PCR). Samples of 37 patients were prospectively evaluated. An internal control was created and applied to sPCR to exclude false-negative results. In total, 21 patients (57%) developed active cytomegalovirus infection. After analyzing the two methods for the diagnosis of active infection, higher sensitivity and negative predictive value of the L-PCR versus sPCR (100% versus 62%), and higher specificity and positive predictive value of sPCR versus L-PCR (81% versus 50% and 72%, respectively) were observed. Discordant results were observed in 11 patients who were L-PCR-positive but sPCR-negative for active cytomegalovirus infection, five of whom developed clinical symptoms of cytomegalovirus. Clinical symptoms were observed in 14 patients, 12 of whom were diagnosed with active infection by nested L-PCR (P=0.007) and seven by nested sPCR (P=0.02). Higher specificity and a positive predictive value for sPCR were observed. Nested L-PCR and sPCR were considered to be complementary methods for the diagnosis and management of symptomatic cytomegalovirus infection.24e69-7

Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Flavonoids, coumarins and other polyphenolic compounds are powerful antioxiants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. Despite being widely used as powerful therapeutic agents for blood coagulation disorders, more specifically to control some serine protease enzymes, the mechanism of anti-inflammatory activity of coumarins is unknown, unlike that of flavonoids. Although their controlling effect on serine proteases is well acknowledged, their action on secretory phospholipase A2 (sPLA2) remains obscure. The present study describes the interaction between umbelliferone (7-HOC) and the sPLA2 from Crotalus durissus collilineatus venom. In vitro inhibition of sPLA2 enzymatic activity by 7-HOC was estimated using 4N3OBA as substrate, resulting in an irreversible decrease in such activity proportional to 7-HOC concentration. The biophysical interaction between 7-HOC and sPLA2 was examined by fluorescent spectral analysis and circular dichroism studies. Results from both techniques clearly showed that 7-HOC strongly modified the secondary structure of this enzyme and CD spectra revealed that it strongly decreased sPLA2 alphahelical conformation. In addition, two-dimensional electrophoresis indicated an evident difference between HPLC-purified native and 7-HOC-treated sPLA2s, which were used in pharmacological experiments to compare their biological activities. In vivo anti-inflammatory activity was assessed by the sPLA2-induced mouse paw edema model, in which 7-HOC presented an effect similar to those of dexamethasone and cyproheptacline against the pro-inflammatory effect induced by native sPLA2 on the mouse paw edema, mast cell degranulation and skin edema. On the other hand, 7-HOC exhibited a more potent inhibitory effect on sPUL2 than that of p-bromophenacyl bromide (p-BPB). Our data suggest that 7-HOC interacts with sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this enzyme, indicating its potential use to suppress inflammation induced by sPLA2 from the snake venom. (C) 2008 Published by Elsevier Ltd.534417426Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [06/55778-2, 07/54714-3

Peripheral blood leukocytes and serum nested polymerase chain reaction are complementary methods for monitoring active cytomegalovirus infection in transplant patients

BACKGROUND: Human cytomegalovirus is an important cause of morbidity and mortality in immunocompromised patients. Qualitative polymerase chain reaction (PCR) has proven to be a sensitive and effective technique in defining active cytomegalovirus infection, in addition to having low cost and being a useful test for situations in which there is no need for quantification. Real-time PCR has the advantage of quantification; however, the high cost of this methodology makes it impractical for routine use. OBJECTIVE: To apply a nested PCR assay to serum (sPCR) and to evaluate its efficiency to diagnose active cytomegalovirus infection compared with PCR of peripheral blood leukocytes (L-PCR). METHODS: Samples of 37 patients were prospectively evaluated. An internal control was created and applied to sPCR to exclude false-negative results. RESULTS: In total, 21 patients (57%) developed active cytomegalovirus infection. After analyzing the two methods for the diagnosis of active infection, higher sensitivity and negative predictive value of the L-PCR versus sPCR (100% versus 62%), and higher specificity and positive predictive value of sPCR versus L-PCR (81% versus 50% and 72%, respectively) were observed. Discordant results were observed in 11 patients who were L-PCR-positive but sPCR-negative for active cytomegalovirus infection, five of whom developed clinical symptoms of cytomegalovirus. Clinical symptoms were observed in 14 patients, 12 of whom were diagnosed with active infection by nested L-PCR (P= 0.007) and seven by nested sPCR (P= 0.02). Higher specificity and a positive predictive value for sPCR were observed. CONClUSION: Nested L-PCR and sPCR were considered to be complementary methods for the diagnosis and management of symptomatic cytomegalovirus infection.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.243E69E7

Peripheral Blood Leukocytes and Serum Nested Polymerase Chain Reaction Are Complementary Methods for Monitoring Active Cytomegalovirus Infection in Transplant Patients

BACKGROUND: Human cytomegalovirus is an important cause of morbidity and mortality in immunocompromised patients. Qualitative polymerase chain reaction (PCR) has proven to be a sensitive and effective technique in defining active cytomegalovirus infection, in addition to having low cost and being a useful test for situations in which there is no need for quantification. Real-time PCR has the advantage of quantification; however, the high cost of this methodology makes it impractical for routine use

A Catalytically Inactive Lys49 PLA2 Isoform from Bothrops jararacussu venom that Stimulates Insulin Secretion in Pancreatic Beta Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)A new secretory phospholipase A2 (sPLA2) isoform from Bothrops jararacussu venom (BjVIII) has been characterized by causing platelet aggregation, an absent activity in BthTx-I, Prtx-I and PrTx-II sPLA2s. According to our results, BjVIII also enhances insulin release by the pancreatic beta cells. The complete amino acid sequence of the new isoform was determined by Edman degradation and de novo peptide sequencing. These analyses showed a G35K amino acid modification for BjVIII in comparison with BthTx-I, PrTx-I and Prtx-II, a structural difference that has been related to the conflicting biological activities among BjVIII and other Lys49 sPLA2s. The whole set of evidences collected in this work indicates that, besides the C-terminal region and B-wing of PLA2, the calcium binding loop in BjVIII should be considered as an important region, involved in the pharmacological effects of Lys49-sPLA2 isoforms from the Bothrops genus.181111331139Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2007/54714-3]CNPq [301665/2007-9

Restriction Fragment Length Polymorphism of Urease C and Urease B Genes of Helicobacter pylori Strains Isolated from Brazilian Patients with Peptic Ulcer and Chronic Gastritis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The aim of the present work is to identify the presence of Helicobacter pylori bacterium in samples of gastric mucosa fragments, obtained by gastric biopsy, from Brazilian patients with peptic ulcer and chronic gastritis and also to determine differences among the prevalent strains in these two diseases by urease C and urease B genes amplification utilizing nested polymerase chain reaction (PCR) and PCR. We encountered 17 genotyping patterns for urease C and 7 for urease B and, although no significant differences were found among the patterns encountered for both diseases, we found predominant groups for each disease. Typing methods of the products obtained by nested PCR and PCR show a functional scheme and are of great importance for epidemiologic studies and H. pylori strain characterization, in addition to allowing correlation among the several strains and their role in the diseases caused by this microorganism.54714871493Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34 Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitre-3-(octanoyloxy)benzoic acid, determined its V(max) (7.56 nmoles/min) and K(M) (2.76 mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75 mM and 0.4 mu M, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2. (c) 2008 Elsevier Ltd. All rights reserved.531104114FundaiAo de Atnaparo a Pescluisa do Estado de SAO Paulo/Proc [06/55778-2]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Ao de Atnaparo a Pescluisa do Estado de SAO Paulo/Proc [06/55778-2]CNPq [301665/2007-9