Heparin is biocompatible and can induce differentiation of human dental pulp cells.

AIM: To investigate the biocompatibility, osteogenic bioactivity and mRNA expression of the osteo/odontogenic markers bone morphogenetic protein 2 (BMP-2), osteocalcin (OC) and alkaline phosphatase (ALP), induced by heparin in human dental pulp cells (hDPCs). METHODOLOGY: hDPCs were exposed to the heparin, and cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT), and cell death was evaluated by flow cytometry. Osteogenic bioactivity was evaluated by the alkaline phosphatase (ALP) assay, and the detection of calcium deposits by alizarin red staining (ARS). The gene expression of BMP-2, OC and ALP was quantified with real-time PCR. Statistical analysis was performed with ANOVA and Bonferroni or Tukey post-test and t-test (α = 0.05). RESULTS: Heparin had no cytotoxic effect and did not induce apoptosis. After 3 days, heparin had significantly higher ALP activity in comparison with the control (P < 0.05). Heparin had a significant (P < 0.05) stimulatory effect on the formation of mineralized nodules. BMP-2 and OC mRNA expressions were significantly higher in cells exposed to heparin than control group after 1 day (P < 0.05). CONCLUSIONS: Heparin was biocompatible in hDPCs, induced osteogenic bioactivity and enhanced mRNA expression of osteo/odontogenic markers BMP-2 and OC. These results suggest that heparin has potential to induce osteo/odontogenic cell differentiation of hDPCs.

Inga edulis fruits: a new source of bioactive anthocyanins.

The extraction conditions and chromatographic analysis from seeds of Inga edulis were optimized and provided one anthocyanin from aqueous fraction and a mixture of three anthocyanins from methanolic fraction. The pure anthocyanin obtained was subjected to structural modifications and the products obtained were subjected to chemical and pharmacological assays, as well as quantum chemical calculations based on DFT and TD-DFT methods. Hence, the anthocyanin fractions were evaluated for their chemical-pharmacological potential through chemical and biological assays: antioxidant activity by the DPPH, determination of the Solar Protection Factor (SPF) and cytotoxic activity (hepatocellular carcinoma infected with hepatitis C virus). The results indicated that even the anthocyanin and derivatized compounds having high antioxidant potential showed an SPF lower than six, which is lower than the minimum accepted by current Brazilian legislation. In addition, none of compounds presented significant cytotoxic activity against the tumour cell line studied.

CCR5 genotype and plasma beta-chemokine concentration of Brazilian HIV-infected individuals.

The 32-bp deletion in the HIV-1 co-receptor CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals for the deleted allele and partial protection against HIV-1 during disease progression in heterozygotes. Natural ligands for CCR5, MIP-1alpha, MIP-1beta and RANTES, have been shown to inhibit HIV replication in CD4+ T cells. In the present study, we examined the CCR5 genotype by PCR and the plasma levels of RANTES and MIP-1alpha by ELISA among blood donors (N = 26) and among HIV-1-infected individuals (N = 129). The control group consisted of healthy adult volunteers and HIV-1-infected subjects were an asymptomatic and heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. The frequency of the CCR5 mutant allele (Delta32ccr5) in this population was 0.032; however, no Delta32ccr5 homozygote was detected. These results could be related to the intense ethnic admixture of the Brazilian population. There was no correlation between circulating beta-chemokines (MIP-1alpha, RANTES) and viral load in HIV-infected individuals. RANTES concentrations in plasma samples from HIV+ patients carrying the homozygous CCR5 allele (CCR5/CCR5) (28.23 ng/ml) were higher than in the control samples (16.07 ng/ml; P<0.05); however, this HIV+ patient group (mean 26.23 pg/ml) had significantly lower concentrations of MIP-1alpha than those observed in control samples (mean 31.20 pg/ml; P<0.05). Both HIV-1-infected and uninfected individuals heterozygous for the Delta32ccr5 allele had significantly lower concentrations of circulating RANTES (mean 16.07 and 6.11 ng/ml, respectively) than CCR5/CCR5 individuals (mean 28.23 and 16.07 ng/ml, respectively; P<0.05). These findings suggest that the CCR5 allele and beta-chemokine production may affect the immunopathogenesis of HIV-1.

Citrus and Coffee Strains of Xylella fastidiosa Induce Pierce's Disease in Grapevine.

Xylella fastidiosa causes citrus variegated chlorosis (CVC) disease in Brazil and Pierce's disease of grapevines in the United States. Both of these diseases cause significant production problems in the respective industries. The recent establishment of the glassy-winged sharpshooter in California has radically increased the threat posed by Pierce's disease to California viticulture. Populations of this insect reach very high levels in citrus groves in California and move from the orchards into the vineyards, where they acquire inoculum and spread Pierce's disease in the vineyards. Here we show that strains of X. fastidiosa isolated from diseased citrus and coffee in Brazil can incite symptoms of Pierce's disease after mechanical inoculation into seven commercial Vitis vinifera varieties grown in Brazil and California. Thus, any future introduction of the CVC strains of X. fastidiosa into the United States would pose a threat to both the sweet orange and grapevine industries. Previous work has clearly shown that the strains of X. fastidiosa isolated from Pierce's disease- and CVC-affected plants are the most distantly related of all strains in the diverse taxon X. fastidiosa. The ability of citrus strains of X. fastidiosa to incite disease in grapevine is therefore surprising and creates an experimental system with which to dissect mechanisms used by X. fastidiosa in plant colonization and disease development using the full genome sequence data that has recently become available for both the citrus and grapevine strains of this pathogen.

Coffee Leaf Scorch Caused by a Strain of Xylella fastidiosa from Citrus.

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var. robusta 'Apuatão 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosa and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X. fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

[Immunological aspects of the phenoloxidase enzymatic system of Schistosoma mansoni]. / Aspectos imunológicos do sistema enzimático fenoloxidase de Schistosoma mansoni.

The phenol oxidase enzymatic system (EC 1.10.3.1, EC 1.10.3.2) is widespread in different species of the animal and vegetal kingdom. Despite its importance in the eggshell formation of the trematodes phenol oxidase (PO) has been little studied in these organisms, mainly in S. mansoni. This report presents the initial results concerning the immunization of rabbits with PO of S. mansoni and mushroom tyrosinase. The immunological analysis done by means of double immunodifusion (Ouchterlony) and immunoelectrophoresis techniques revealed some immunological identity between the PO of males and females. It was not seen cross reaction between the antisera against PO and tyrosinase, what suggests that the antigenic determinants of both enzymes are different in spite of their catalytic sites being similar, since they act over the same substrate. The results reported here represent a first step in way to obtain the PO isoenzymes in their pure form and should open new insights for further studies on the molecular mechanisms involved in the sclerotization process of the S. mansoni eggshell.

South American rattlesnake venom: its hemolytic power.

The hemolytic power of rattlesnake venom (Crotalus durissus terrificus) was studied. A high percentage of sample with negative hemolytic power was detected when sheep red blood cells were used. A large number of venoms with hemolytic power, though with a low hemolysis percentage, were detected when liquid, recently extracted venom was used. When crystallized venom was used under the same experimental conditions, a higher percentage of positivity for hemolysis was obtained. When the results obtained on agar plates were compared to those obtained in test tubes, a large number of animals with a higher percentage of hemolysis were detected, though this value was not proportional to the number of animals showing positive plate hemolysis. When the hemolytic power of these venoms was tested on human red blood cells, a large percentage of animals with venoms having a low hemolytic power was also detected. Hemolytic power was much greater when human red blood cells were tested with crystallized venom. The preparation of red blood cells also had an important effect and the use of red blood cells from defibrinated blood is recommended. We conclude that rattlesnake venom has hemolytic power that increases when the venom is crystallized. Red blood cells should be properly prepared for the lysis reactions. We suggest that the lytic power of the venom is related to venom concentration and to the purity of its fractions.

Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein.

In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L(-1) of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and confirmed by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1:10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1:1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application.

Polymorphism of the ITS-2 region of the ribosomal DNA of the Triatominae Rhodnius domesticus, R. pictipes, R. prolixus and R. stali.

The polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP) was used to compare Rhodnius domesticus (Neiva & Pinto), R. pictipes (Stal), R. prolixus (Stal) and R. stali (Lent, Jurberg & Galvão) (Hemiptera: Reduviidae). The enzyme BstUI differentiated R. domesticus, R. pictipes and R. prolixus, and HhaI differentiated R. domesticus, R. pictipes and R. stali. With the fingerprinting analysis generated by these two enzymes, it was possible to clearly identify all four species in the study.

Rattlesnake venom: action upon erythrocytes and leucocytes of rats.

We studied the action of whole rattlesnake venom on red blood cells and leucocytes of adult male and female rats. Animals were surgically cannulated for blood collection directly from the inferior caval vein and injected intramuscularly in the thigh with a mixture of venoms from a large number of rattlesnakes. The signs shown by the animals were paralysis of the hind part of the body, lack of motor coordination, and respiratory difficulties, with death occurring in some cases. Necroscopy showed petechial hemorrhage in the intestine and jejunum and darkening of the viscera, which was found to be due to engorged blood vessels upon histopathological examination. Blood examination showed a change in color to dark brown due to the transformation of hemoglobin to methemoglobin. Venom fractions were found to have a low hemolytic power because of their low concentration in the venom samples. Blood sedimentation rate showed a clear variation, especially 60 minutes after venom injection. Both phenomena may be linked to the lytic power of the venoms. An interesting phenomenon was that the animals showed initial leucopenia, which was followed by persistent leucocytosis. Lymphocytopenia and increased neutrophil numbers were also observed. The present results led us to conclude that rattlesnake venom has a relative hemolytic power which increases with venom concentration and with the concentration of the fractions in whole venom.

Strains of Xylella fastidiosa rapidly distinguished by arbitrarily primed-PCR.

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0. 872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors.

CCR5 genotype and plasma ß-chemokine concentration of Brazilian HIV-infected individuals

The 32-bp deletion in the HIV-1 co-receptor CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals for the deleted allele and partial protection against HIV-1 during disease progression in heterozygotes. Natural ligands for CCR5, MIP-1alpha, MIP-1ß and RANTES, have been shown to inhibit HIV replication in CD4+ T cells. In the present study, we examined the CCR5 genotype by PCR and the plasma levels of RANTES and MIP-1alpha by ELISA among blood donors (N = 26) and among HIV-1-infected individuals (N = 129). The control group consisted of healthy adult volunteers and HIV-1-infected subjects were an asymptomatic and heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. The frequency of the CCR5 mutant allele (delta32ccr5) in this population was 0.032; however, no delta32ccr5 homozygote was detected. These results could be related to the intense ethnic admixture of the Brazilian population. There was no correlation between circulating ß-chemokines (MIP-1alpha, RANTES) and viral load in HIV-infected individuals. RANTES concentrations in plasma samples from HIV+ patients carrying the homozygous CCR5 allele (CCR5/CCR5) (28.23 ng/ml) were higher than in the control samples (16.07 ng/ml; P<0.05); however, this HIV+ patient group (mean 26.23 pg/ml) had significantly lower concentrations of MIP-1alpha than those observed in control samples (mean 31.20 pg/ml; P<0.05). Both HIV-1-infected and uninfected individuals heterozygous for the delta32ccr5 allele had significantly lower concentrations of circulating RANTES (mean 16.07 and 6.11 ng/ml, respectively) than CCR5/CCR5 individuals (mean 28.23 and 16.07 ng/ml, respectively; P<0.05). These findings suggest that the CCR5 allele and ß-chemokine production may affect the immunopathogenesis of HIV-1

Fator de crescimento derivado de plaquetas humanas obtido por uma metodologia rápida e de baixo custo / Human platelet-derived growth factor obtained by means of a fast and inexpensive methodology

O fator de crescimento derivado de plaquetas (PDGF) é uma proteína catiônica armazenado principalmente nos grânulos-alfa plaquetário. O PDGF por ser altamente mitogênico, principalmente para fibroblastos e leiomiócitos, é de grande importância no processo de regeneração tecidual. Neste trabalho, o PDGF foi obtido a partir do lisado de plaquetas humanas (5 x 10 plaquetas/mL) vencidas, purificado por cromatografia de troca iônica em resina de CM-Sepharose. A proteína eluída na fração catiônica, foi identificada por anticorpos policlonais anti-PDGF (AA e BB) e, posteriormente, sua atividade mitogênica confirmada pela incorporação da [H3]-TdR usando fibroblastos da linhagem celular NIH/3T3 em cultura. A comparação entre os efeitos mitogênicos sobre o PDGF, fração catiônica, com uma proteína recombinante controle (PDGF-AB), revelou que a proteína parcialmente purificada induziu semelhante estimulação, 157.567 cpm e 165.796 cpm, respectivamente. Os resultados obtidos evidenciam a aplicabilidade dessas condições experimentais na obtenção do PDGF, preservando sua atividade biológica, através de procedimentos de baixa complexibilidade, eficiente e de custo operacional reduzido, possibilitando seu uso em experimentos de regeneração tecidual

Rattlesnake venom: action upon erythrocytes and leucocytes of rats

Hemos estudiado la acción del veneno completo de serpiente de cascabel sobre los glóbulos rojos y leucocitos de ratas adultas machos y hembras. Los animales fueron quirúrgicamente canulados para realizar recolecciones de sangre directamente de la vena cava inferior, e inyectados intramuscularmente en el muslo con la mezcla de veneno obtenido de un gran número de cascbeles. Los signos mostrados por los animales fueron parálisis de la parte posterior del cuerpo, déficit de la coordinación motora y dificultad repiratoria, con muerte en algunos casos. La autopsia mostró hemorragias petequiales en el intestino y yeyuno, además de oscurecimiento visceral y obstrucción de los vasos sanguíneos verificada en el examen histopatológico. Los exámenes sanguíneos mostrarón cambios de color hacia marrón oscuro debido a la transformación de la hemoglobina en metahemoglobina. Los índices de sedimentación sanguínea mostraron una clara variación, especialmente 60 minutos después de inuectado el veneno. Ambos fenómenos pueden estar ligados al poder lítico de los venenos. Un interesante fenómeno fue el que los animales presentaron inicialmente leucopenia, que fue seguida por persistente leucocitosis. Los presentes reultados nos llevan a concluir que el veneno de cascabel tiene un relativo poder hemolítico que se incrementa con la concentración del veneno y con la concentración de las fracciones en veneno completo (AU)

Rattlesnake venom: action upon erythrocytes and leucocytes of rats

Hemos estudiado la acción del veneno completo de serpiente de cascabel sobre los glóbulos rojos y leucocitos de ratas adultas machos y hembras. Los animales fueron quirúrgicamente canulados para realizar recolecciones de sangre directamente de la vena cava inferior, e inyectados intramuscularmente en el muslo con la mezcla de veneno obtenido de un gran número de cascbeles. Los signos mostrados por los animales fueron parálisis de la parte posterior del cuerpo, déficit de la coordinación motora y dificultad repiratoria, con muerte en algunos casos. La autopsia mostró hemorragias petequiales en el intestino y yeyuno, además de oscurecimiento visceral y obstrucción de los vasos sanguíneos verificada en el examen histopatológico. Los exámenes sanguíneos mostrarón cambios de color hacia marrón oscuro debido a la transformación de la hemoglobina en metahemoglobina. Los índices de sedimentación sanguínea mostraron una clara variación, especialmente 60 minutos después de inuectado el veneno. Ambos fenómenos pueden estar ligados al poder lítico de los venenos. Un interesante fenómeno fue el que los animales presentaron inicialmente leucopenia, que fue seguida por persistente leucocitosis. Los presentes reultados nos llevan a concluir que el veneno de cascabel tiene un relativo poder hemolítico que se incrementa con la concentración del veneno y con la concentración de las fracciones en veneno completo

Rattlesnake venom: action upon erythrocytes and leucocytes of rats.

We studied the action of whole rattlesnake venom on red blood cells and leucocytes of adult male and female rats. Animals were surgically cannulated for blood collection directly from the inferior caval vein and injected intramuscularly in the thigh with a mixture of venoms from a large number of rattlesnakes. The signs shown by the animals were paralysis of the hind part of the body, lack of motor coordination, and respiratory difficulties, with death occurring in some cases. Necroscopy showed petechial hemorrhage in the intestine and jejunum and darkening of the viscera, which was found to be due to engorged blood vessels upon histopathological examination. Blood examination showed a change in color to dark brown due to the transformation of hemoglobin to methemoglobin. Venom fractions were found to have a low hemolytic power because of their low concentration in the venom samples. Blood sedimentation rate showed a clear variation, especially 60 minutes after venom injection. Both phenomena may be linked to the lytic power of the venoms. An interesting phenomenon was that the animals showed initial leucopenia, which was followed by persistent leucocytosis. Lymphocytopenia and increased neutrophil numbers were also observed. The present results led us to conclude that rattlesnake venom has a relative hemolytic power which increases with venom concentration and with the concentration of the fractions in whole venom.

Doença arterial coronariana: um novo paradigma na AIDS / Coronary artery disease: a new paradigm in AIDS

A inclusão de indivíduos portadores da infecção pelo HIV num grupo propenso às altraçòes metabólicas, fortalece a hipótese de que a AIDS contribui como um fator de risco para o desenvolvimento de desordens vaso-oclusivas. O curso e a progressão da AIDS, bem como as medidas terapêuticas contra o HIV têm sido capazes de mostrar uma infinidade de alterações metabólicas aos quais os portadores estão sujeitos. Esses transtornos afetam o metabolismo de componentes plasmáticos como os lipídeos e a homocisteína e tem sido verificado nos portadores do HIV como consequênciade três fatores predominantemente: (i) a própria infecção viral per se que altera os níveis de lipídeos plasmáticos; (ii) deficiências vitamínicas e de micronutrientes, favorecendo a hiper-homocisteinemia (iii) a terapia medicamentosa antiretroviral, que acompanha efeitos idiossincráticos no metabolismo lipídico do portador. Nesse contexto, o indivíduo infectado pelo HIV está inserido num rol de anormalidades que são fatores de risco para a aterogênese. Essas observações podem ser valiosas quando se verifica que, se a sobrevida do portador está sendo aumentada por ocasião da terapia mais efetiva, é por outro lado possível, que seu risco de desenvolver alteraçòes metabólicas e, portanto, iniciar um processo aterogênico, ou exacerbar algum preexistente, tenha também aumento proporcional