P-glycoprotein and multidrug resistance-associated protein-1 expression in acute myeloid leukemia: Biological and prognosis implications.

BACKGROUND: Despite the advances in the cure rate for acute myeloid leukemia (AML), a considerable number of patients die from the disease due to the occurrence of multidrug resistance (MDR). Overexpression of the transporter proteins, such as P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP), confers resistance to the treatment of these leukemias. METHODS: To analyze the expression of the Pgp and MRP1 in patients with AML and determine their correlation between expression and demographic, clinical, and laboratorial variables, bone marrow and peripheral blood samples from 346 patients with a diagnosis of AML were assessed for the expression of Pgp and MRP1 by flow cytometry. RESULTS: The expression of Pgp and MRP1 was found in 111 (32.1%) and 133 (38.4%) patients, respectively, with greater prevalence in older patients and lower in children, while also observing a high incidence in patients with refractory, recurrence, and secondary disease in comparison with the cases of de novo AML. Regarding the laboratory findings, we observed an association between the expression of Pgp and MRP1 and CD34, CD7, and also M7, M5a, and M2-AML of French-American-British classification. CONCLUSIONS: The results showed that the detection of MDR phenotype by flow cytometry can be a molecular marker for prognosis of patients with AML.

In vitro anticoagulant and antioxidant activities of Jatropha gossypiifolia L. (Euphorbiaceae) leaves aiming therapeutical applications.

BACKGROUND: Jatropha gossypiifolia L. (Euphorbiaceae) is a medicinal plant largely used in folk medicine. Teas from the leaves are popularly used as an antithrombotic agent and the branches are frequently employed as a "thick blood" agent. Considering that the anticoagulant activity associated with antioxidant properties could be beneficial for various cardiovascular diseases, this study's aim is the evaluation of anticoagulant and antioxidant activities of J. gossypiifolia leaves, seeking new therapeutic purposes for this plant. METHODS: The aqueous leaf crude extract (CE) was prepared by decoction and was fractionated by liquid-liquid partition with solvents of increasing polarity. The phytochemical analysis was performed by thin layer chromatography (TLC) and by the spectrophotometric quantification of sugars, proteins and phenolic compounds. The anticoagulant activity was evaluated by prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests. The capacity to act in the fibrinolytic system (fibrinolytic and fibrinogenolytic activities) was also assessed. The antioxidant activity was evaluated by total antioxidant capacity, reducing power, copper chelating activity, iron chelating activity, hydroxyl radical scavenging activity and superoxide radical scavenging assays. The potential toxicity was evaluated using hemolytic assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay on HEK-293 cells. RESULTS: CE showed significant anticoagulant activity in aPTT test, while no action was observed in PT test, suggesting a preferential action toward the intrinsic and/or common pathway of coagulation. No effect was observed in the fibrinolytic system. Using the aPTT test, it was observed that the residual aqueous (RA) fraction was the most active, being two times more active than CE. RA presented very significant antioxidant activity in all models tested comparable to or even higher than CE. Regarding the safety, CE and RA did not produce significant cytotoxicity in both tests employed. Phytochemical analysis revealed the presence of alkaloids, flavonoids, proteins, tannins, steroids and/or terpenoids and sugars. CONCLUSIONS: CE and RA possessed significant anticoagulant and antioxidant activity and absence of cytotoxic effect in vitro, thus showing the potential of the plant, especially RA fraction, as a new source of bioactive molecules for therapeutic purposes, with particular emphasis on the treatment of cardiovascular diseases.

Aqueous leaf extract of Jatropha gossypiifolia L. (Euphorbiaceae) inhibits enzymatic and biological actions of Bothrops jararaca snake venom.

Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A2 (Lys49 PLA2). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.

Protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves: enzymatic characterization and procoagulant and fibrinogenolytic activities.

Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

Determination of ß haplotypes in patients with sickle-cell anemia in the state of Rio Grande do Norte, Brazil.

ß(S) haplotypes were studied in 47 non-related patients with sickle-cell anemia from the state of Rio Grande do Norte, Brazil. Molecular analysis was conducted by PCR/RFLP using restriction endonucleases XmnI, HindIII, HincII and HinfI to analyze six polymorphic sites from the beta cluster. Twenty-seven patients (57.5%) were identified with genotype CAR/CAR, 9 (19.1%) CAR/BEN, 6 (12.8%) CAR/CAM, 1 (2.1%) BEN/BEN, 2 (4.3%) CAR/Atp, 1 (2.1%) BEN/Atp and 1 (2.1%) with genotype Atp/Atp. The greater frequency of Cameroon haplotypes compared to other Brazilian states suggests the existence of a peculiarity of African origin in the state of Rio Grande do Norte.

Characterization of beta-thalassemia mutations in patients from the state of Rio Grande do Norte, Brazil.

35 unrelated individuals were studied for characterization as either heterozygous or homozygous for beta-thalassemia. Molecular analysis was done by PCR/RFLP to detect the mutations most commonly associated with beta-thalassemia (ß(0)IVS-I-1, ß(+)IVS-I-6, and ß(0)39). In the patients who showed none of these mutations, the beta-globin genes were sequenced. Of the 31 heterozygous patients, 13 (41.9%) had the ß(+)IVS-I-6 mutation, 15 (48.4%) the ß(0)IVS-I-1 mutation, 2 (6.5%) the ß(+)IVS-I-110 mutation and 1 (3.2%) the ß(+)IVS-I-5 mutation. IVS-I-6 was detected in the four homozygotes. The mutation in codon 39, often found in previous studies in Brazil, was not detected in the present case. This is the first study aiming at identifying mutations that determine beta-thalassemia in the state of Rio Grande do Norte.

Determination of beta S haplotypes in patients with sickle-cell anemia in the state of Rio Grande do Norte, Brazil

βS haplotypes were studied in 47 non-related patients with sickle-cell anemia from the state of Rio Grande do Norte, Brazil. Molecular analysis was conducted by PCR/RFLP using restriction endonucleases XmnI, HindIII, HincII and HinfI to analyze six polymorphic sites from the beta cluster. Twenty-seven patients (57.5 percent) were identified with genotype CAR/CAR, 9 (19.1 percent) CAR/BEN, 6 (12.8 percent) CAR/CAM, 1 (2.1 percent) BEN/BEN, 2 (4.3 percent) CAR/Atp, 1 (2.1 percent) BEN/Atp and 1 (2.1 percent) with genotype Atp/Atp. The greater frequency of Cameroon haplotypes compared to other Brazilian states suggests the existence of a peculiarity of African origin in the state of Rio Grande do Norte.

Characterization of beta-thalassemia mutations in patients from the state of Rio Grande do Norte, Brazil

35 unrelated individuals were studied for characterization as either heterozygous or homozygous for beta-thalassemia. Molecular analysis was done by PCR/RFLP to detect the mutations most commonly associated with beta-thalassemia (β0IVS-I-1, β+IVS-I-6, and β039). In the patients who showed none of these mutations, the beta-globin genes were sequenced. Of the 31 heterozygous patients, 13 (41.9 percent) had the β+IVS-I-6 mutation, 15 (48.4 percent) the β0IVS-I-1 mutation, 2 (6.5 percent) the β+IVS-I-110 mutation and 1 (3.2 percent) the β+IVS-I-5 mutation. IVS-I-6 was detected in the four homozygotes. The mutation in codon 39, often found in previous studies in Brazil, was not detected in the present case. This is the first study aiming at identifying mutations that determine beta-thalassemia in the state of Rio Grande do Norte.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment.

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. One hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p<0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p<0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p=0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4+/-12.4%) and apolipoprotein B (apoB) (17.0+/-31.3%) when compared with the higher quartile (>0.085: LDL-c=40.3+/-14.3%; apoB=32.5+/-10.7%; p<0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphismsand lowering-cholesterol response. One hundred and thirty-six individuals withhypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) ( p 0.085: LDL-c = 40.3 14.3%; apoB = 32.5 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversedthe effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin.

CYP3A53A allele is associated with reduced lowering-lipid response to atorvastatin in individuals with hypercholesterolemia.

BACKGROUND: The cytochrome P450 isoenzyme 3A5 (CYP3A5) has an important role on biotransformation of xenobiotics. CYP3A5 SNPs have been associated with variations on enzyme activity that can modify the metabolism of several drugs. METHODS: In order to evaluate the influence of CYP3A5 variants on response to lowering-cholesterol drugs, 139 individuals with hypercholesterolemia were selected. After a wash-out period of 4 weeks, individuals were treated with atorvastatin (10 mg/day/4 weeks). Genomic DNA was extracted by a salting-out procedure. CYP3A5*3C, CYP3A5*6 and CYP3A5*1D were analyzed by PCR-RFLP and DNA sequencing. RESULTS: >Frequencies of the CYP3A5*3C and CYP3A5*1D alleles were lower in individuals of African descent (*3C: 47.8% and *1D: 55.2%) than in non-Africans (*3C: 84.9% and *1D 84.8%, p<0.01). Non-Africans carrying *3A allele (*3C and *1D combined alleles) had lower total and LDL-cholesterol response to atorvastatin than non-*3A allele carriers (p<0.05). CONCLUSION: CYP3A5*3A allele is associated with reduced cholesterol-lowering response to atorvastatin in non-African individuals.

Effects of ABCA1 SNPs, including the C-105T novel variant, on serum lipids of Brazilian individuals.

BACKGROUND: ABCA1 plays an important role in HDL metabolism. Single nucleotide polymorphisms (SNPs) in ABCA1 gene were associated with variation in plasma HDL-c. METHODS: The effect of the ABCA1 SNPs C-14T, R219K and of a novel variant C-105T on serum lipids was investigated in 367 unrelated Brazilian individuals (224 hypercholesterolemic and 143 normolipidemic). The relation between ABCA1 SNPs and the lipid-lowering response to atorvastatin (10 mg/day/4 weeks) was also evaluated in 141 hypercholesterolemic (HC) individuals. The polymorphisms were detected by PCR-RFLP and confirmed by DNA sequencing. RESULTS: Linkage disequilibrium was found between the SNPs C-105T and C-14T in the HC group. HC individuals carrying -105CT/TT genotypes had higher serum HDL-c and lower triglyceride and VLDL-c concentrations as well as lower TG/HDL-c ratio compared to the -105CC carriers (p<0.05). The R219K SNP was associated with reduced serum triglyceride, VLDL-c and TG/HDL-c ratio in the HC group (p<0.05), and with an increased serum apoAI in NL individuals. The effects of ABCA1 SNPs on basal serum lipids of HC individuals were not modified by atorvastatin treatment. CONCLUSIONS: The ABCA1 SNPs R219K and C-105T were associated with a less atherogenic lipid profile but not with the lowering-cholesterol response to atorvastatin in a Brazilian population.

A novel 3-base deletion (IVS3+2_4delTGG) of the hydroxymethylbilane synthase gene in a Brazilian patient with acute intermittent porphyria

Acute intermittent porphyria (AIP, OMIM 176000) is an autosomal dominant metabolic disease caused by mutations in the gene encoding hydroxymethylbilane synthase (HMBS; EC 4.3.1.8; formely named porphobilinogen deaminase, PBGD), mapped to chromosome 11q23.3. We describe a novel mutation of the HMBS gene, a de novo 3-base deletion in the splicing donor site of intron 3 (IVS3+2_4delTGG) in a woman affected by AIP. RT-PCR analysis revealed an abnormal HMBS mRNA, compatible with exon 3 skipping.

Changes in hematological, hemostatic and biochemical parameters induced experimentally in rabbits by Loxosceles gaucho spider venom.

Human accidents caused by Loxosceles spiders may result in local dermal necrosis and, in some cases, severe systemic reactions - such as intravascular hemolysis, disseminated intravascular coagulation (DIC), renal failure and death. Since many aspects of envenomation by Loxosceles spiders remain unclear, we studied the hematological and hemostatic responses induced by the i.d. injection of 10 microg/kg Loxosceles gaucho venom in rabbits. For this purpose, total blood cell count, platelet function, coagulation tests and biochemical parameters were analysed at 3, 24, 48, 72 and 120 hours after venom administration. Thrombocytopenia and leukopenia were noted at 3 and 24 hours. Histopathological analysis of the skin lesion, performed at 24 hours after venom administration, showed a massive presence of leukocytes and platelets, hemorrhage and thrombus formation at the injection site. At 72 and 120 hours, neutrophilic leukocytosis and thrombocytosis were observed. Platelet hyperaggregation was noticeable at 48 and 72 hours. Haptoglobin and fibrinogen levels were elevated early and remained in high levels over time. Significant increases in coagulation factors V, VII, VIII, IX, X and XI were noted at 120 hours. The results showed that neither intravascular hemolysis nor DIC occurred. However, the early onset of thrombocytopenia and leukopenia are important findings that may be related to dermal necrosis formation during loxoscelism.

Estudo de polimorfismos genéticos da família ABC: efeitos na expressão gênica e na resposta farmacológica a atorvastatina / Genetic polymorphisms study of the ABC family: effects in the gene expression and in the pharmacological response to atorvastatin

As proteínas da ABC são uma família de transportadores de membrana que participam no transporte de lipídeos e na biodisponibilidade de drogas. Alterações em genes dessas proteínas também têm sido associadas a diferenças na resposta a vários fármacos. Neste estudo, foram avaliados os efeitos dos polimorfismos dos genes MDR1 (C3435T e G2677T/A) e MRP1 (G2012T) na expressão de RNAm e concentrações séricas dos lipídeos, em 76 indivíduos com hipercolesterolemia primária, tratados com atorvastatina (10mg/dia/4sem). DNA e RNA foram obtidos a partir de células mononucleares do sangue perférico. Os polimorfismos genéticos foram analisados por PCR-RFLP e a expressão de RNAm foi quantificada por RT-PCR...