Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating Protein, PyAG1

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.

The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity

Recent work has demonstrated that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron-transport chain triggers several pathways of injury [(protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product formation (AGE)] involved in the pathogenesis of diabetic complications by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. On the other hand, PARP activation results in inhibition of GAPDH by poly-ADP-ribosylation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC and AGE formation are prevented by inhibition of PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in rodent models of cardiomyopathy, nephropathy, neuropathy, and retinopathy. PARP activation is also present in microvasculature of human diabetic subjects. The present review focuses on the role of PARP in diabetic complications and emphasizes the therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.

Changes in NAD/ADP-ribose metabolism in rectal cancer

The extent of ADP-ribosylation in rectal cancer was compared to that of the corresponding normal rectal tissue. Twenty rectal tissue fragments were collected during surgery from patients diagnosed as having rectal cancer on the basis of pathology results. The levels of ADP-ribosylation in rectum cancer tissue samples (95.9 ± 22.1 nmol/ml) was significantly higher than in normal tissues (11.4 ± 4 nmol/ml). The level of NAD+ glycohydrolase and ADP-ribosyl cyclase activities in rectal cancer and normal tissue samples were measured. Cancer tissues had significantly higher NAD+ glycohydrolase and ADP-ribosyl cyclase activities than the control tissues (43.3 ± 9.1 vs 29.2 ± 5.2 and 6.2 ± 1.6 vs 1.6 ± 0.4 nmol mg-1 min-1). Approximately 75% of the NAD+ concentration was consumed as substrate in rectal cancer, with changes in NAD+/ADP-ribose metabolism being observed. When [14C]-ADP-ribosylated tissue samples were subjected to SDS-PAGE, autoradiographic analysis revealed that several proteins were ADP-ribosylated in rectum tissue. Notably, the radiolabeling of a 113-kDa protein was remarkably greater than that in control tissues. Poly(ADP)-ribosylation of the 113-kDa protein in rectum cancer tissues might be enhanced with its proliferative activity, and poly(ADP)-ribosylation of the same protein in rectum cancer patients might be an indicator of tumor diagnosis.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 109 infected cells in three liters of culture

Modulation of store-operated Ca2+ entry by cyclic-ADP-ribose

Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70% by several inhibitors of the RyR, including ryanodine (10 µM), dantrolene (10 µM), and ruthenium red (10 µM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 µM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80%. Pre-incubation of cells with 100 µM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 µM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80%. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.

Participação das plaquetas no processo de fibrose dos pacientes com esquistossomose mansônica

O objetivo deste estudo foi avaliar a ativação plaquetária através da P-selectina e o conteúdo de PDGF-AB e TGFbeta1, nos pacientes com esquistossomose que desenvolveram fibrose (F3), naqueles que não tiveram esta manifestação (F0) e nos controles (C). Os resultados mostraram que a percentagem de P-selectina nas plaquetas sem estímulo de agonistas foi de 10,6% nos F3; 11,1% nos FO, e 6,3% nos C e após a adição de ADP/adrenalina, foi de 44%; 25,3% e 42%, respectivamente. A dosagem do PDGF-AB e TGFbeta1 por plaquetas foi de 11,016ng/dL (F3); 3,172 ng/dL (F0) e 5,01ng/dL (C) e, (0,012ng/dL (F3); 5,27ng/dL (F0) e 4,66ng/dL (C), respectivamente. Em relação à P-selectina, concluímos que as plaquetas dos pacientes com esquistossomoses, apesar de estarem pré ativadas, mantiveram-se responsivas aos agonistas. O TFGbeta1 não apresentou diferença entre os três grupos, enquanto o PDGF-AB foi significantemente maior no grupo F3, sugerindo a participação deste no desenvolvimento da fibrose.

Estudo farmacognóstico e atividade in vitro sobre a coagulação sanguínea e agregação plaquetária das folhas de Passiflora nitida Kunth (Passifloraceae)

O gênero Passiflora (Passifloraceae) é utilizado principalmente para tratar doenças do SNC e cardiovasculares. A espécie Passiflora nitida Kunth é comumente conhecida como “maracujá-do-mato". A literatura relata o consumo in natura dos frutos desta espécie pela população local para distúrbios gastrointestinais. Considerando o potencial farmacológico do gênero, este trabalho teve por objetivo realizar estudo de caracterização fitoquímica desta espécie e estudar os efeitos dos extratos aquoso (EA), etanólico (EE) e hexânico (EH) de suas folhas sobre a coagulação sanguínea e agregação plaquetária. Para a caracterização fitoquímica foram realizados testes de cromatografia em camada delgada e ressonância magnética nuclear. O efeito dos extratos sobre a coagulação foi avaliado pelos testes de tempo de protrombina (TP) e tempo de tromboplastina parcial ativada (TTPa). O efeito sobre a agregação plaquetária foi avaliado em plasma rico em plaquetas por método espectrofotométrico, usando adenosina difosfato (ADP) e adrenalina (ADR) como indutores da agregação. Os extratos EA, EE e EH apresentaram atividade coagulante pelo teste do TP e o EE apresentou atividade anticoagulante para o TTPa. Quando induzidos por ADP, os extratos EA, EE e EH apresentaram valores de concentração inibitória 50% (CI50, µg/mL) de 450,5 ± 50,7; 511,2 ± 35,5 e 394,4 ± 8,9, respectivamente, e quando induzidos por ADR apresentaram valores de 438,7 ± 5,2; 21,0 ± 1,9 e 546,9 ± 49,9, respectivamente. O EE apresentou atividade inibitória sobre a agregação. A caracterização fitoquímica foi sugestiva da presença de flavonóides e cumarinas, aos quais podem ser atribuídos, em parte, os efeitos biológicos estudados.

Ausência de interação clopidogrel-estatina em pacientes submetidos a implante de "stent" coronário

FUNDAMENTO: Alguns estudos têm sugerido redução da atividade do clopidogrel sobre a ativação e adesão plaquetárias em pacientes em uso de estatinas. OBJETIVO: Avaliar se a ativação e agregação plaquetárias diminuem com clopidogrel, e se ocorre redução da ação do clopidogrel quando associado à atorvastatina ou à sinvastatina. MÉTODOS: Estudo prospectivo que incluiu 68 pacientes com angina estável em uso prévio de sinvastatina, atorvastatina, ou nenhuma estatina (grupo controle), com indicação prévia eletiva de realização de intervenção coronária percutânea. Foi analisada a ativação plaquetária através do número de plaquetas, níveis de P-selectina e glucoproteína IIb/IIIa (com e sem estímulo de ADP) através de citometria de fluxo. Os resultados foram analisados antes e após a intervenção coronária percutânea e da administração de clopidogrel. RESULTADOS: Observamos redução da atividade plaquetária com uso de clopidogrel. Além disso, não houve diferenças entre as variáveis analisadas que comprovassem redução da atividade do clopidogrel quando associado à estatinas. Observou-se níveis de p-selectina (pré-angioplastia: 14,23±7,52 x 11,45±8,83 x 7,65±7,09; pós angioplastia: 21,49±23,82 x 4,37±2,71 x 4,82±4,47, ρ<0,01) e glicoproteína IIb/IIIa (pré-angioplastia: 98,97±0,43 x 98,79±1,25 x 99,21±0,40; pós angioplastia: 99,37±0,29 x 98,50±1,47 x 98,92±0,88, ρ=0,52), respectivamente nos grupos controle, atorvastatina e sinvastatina. CONCLUSÃO: Concluímos que a ativação plaquetária diminui com a administração de clopidogrel, e que o clopidogrel não tem seu efeito antiplaquetário reduzido na presença de sinvastatina ou atorvastatina.

Prevalência de alta reatividade plaquetária em pacientes tratados com aspirina encaminhados para angiografia coronária

FUNDAMENTO: A aspirina (Ácido Acetilsalicílico - AAS) é capaz de reduzir eventos adversos cardiovasculares em pacientes portadores de Doença Arterial Coronariana (DAC) através da inibição da atividade plaquetária. Alguns pacientes com DAC, apesar da terapia com AAS, apresentam Alta Reatividade Plaquetária (ARP), o que determina um maior risco para o desenvolvimento de eventos cardiovasculares. OBJETIVO: O objetivo deste estudo foi determinar a prevalência de ARP em pacientes tratados com AAS e encaminhados para cinecoronariografia, além de avaliar se existe uma possível correlação entre a gravidade da DAC e o desenvolvimento de ARP. MÉTODOS: Estudo de centro único onde foram incluídos 115 pacientes consecutivos, tratados com AAS e portadores de DAC estável. A reatividade plaquetária induzida pelo ADP e colágeno foram avaliadas através da Agregometria de Transmitância Luminosa (ATL). Pacientes com agregação plaquetária maior que 70%, induzida por ambos os reagentes, foram classificados como tendo ARP e, neste grupo, a adesão ao tratamento com AAS foi avaliada através da dosagem dos níveis séricos de salicilato. RESULTADOS: A média de idade foi de 60,9 anos e a dose média de AAS foi de 164,2 mg. Tabagismo e diabetes melito estavam presentes em 28,7% e 31,5% dos pacientes, respectivamente. Foi encontrada ARP em 14 pacientes (13%), entretanto, em sete indivíduos (50%) com ARP observaram-se baixos níveis séricos de salicilato (< 2,0 µg/mL), sugerindo não adesão à terapia medicamentosa. Em 6,5% dos pacientes com ARP identificou-se níveis detectáveis de salicilato sérico, sugerindo uma eficácia reduzida do AAS. A ARP se correlacionou com o número e a gravidade das estenoses coronárias (p = 0,04). CONCLUSÃO: Em uma população de pacientes tratados com AAS e encaminhados para angiografia coronária, a reatividade plaquetária elevada é prevalente (13%), sendo 50% destes pacientes não aderentes à terapia farmacológica e 50% apresentam redução da efetividade da droga.

Alta reatividade plaquetária durante tratamento é preditora de eventos cardíacos em pacientes com stents farmacológicos

FUNDAMENTO: O papel dos testes de reatividade plaquetária (RP) na predição de eventos em longo prazo em pacientes latino-americanos tratados com stents farmacológicos (SF) não foi estabelecido. OBJETIVOS: Analisar o papel dos testes de RP na predição de eventos após a implantação de SF. MÉTODOS: De maio de 2006 a janeiro de 2008, foram incluídos 209 pacientes brasileiros que se submeteram a tratamento eletivo com SF. A RP foi avaliada 12 a 18 horas após o procedimento, por agregometria de transmitância de luz com 5µM de ADP. Os pacientes foram acompanhados prospectivamente por até 4,8 anos. Dezessete (8%) dos indivíduos foram perdidos durante o acompanhamento e a coorte final foi composta de 192 pacientes. A curva ROC foi utilizada para determinar o melhor ponto de corte de 5µM de ADP para prever eventos. O endpoint primário foi uma combinação de morte cardiovascular, infarto agudo do miocárdio, trombose definitiva de stent, e revascularização de artéria alvo.Modelos de risco proporcional de Cox foram utilizados para determinar as variáveis independentemente associadas com o tempo até o primeiro evento. RESULTADOS: O melhor ponto de corte de 5µM de ADP foi de 33%. Cento e sete (55,7%) pacientes apresentaram 5mM de ADP > 33%. A taxa de sobrevivência livre de eventos em 1.800 dias foi de 55% contra 70% para os indivíduos com ADP5 acima e abaixo desse ponto de corte, respectivamente (p = 0,001). Preditores de tempo independentes para o primeiro evento foram tabagismo atual (HR 3,49, IC95%: 1,76-6,9, p = 0,0003), ADP 5mM > 33% (HR 1,95, IC95%: 1,09-3,51, p = 0,025) e idade (HR 1,03 IC 95%: 1,0-1,06, p = 0,041). CONCLUSÕES: Neste estudo, 55,7% dos pacientes apresentaram alta reatividade plaquetária durante tratamento. 5µM de ADP > 33% foi um preditor independente de eventos em longo prazo.

Fixação do dióxido de carbono por bactérias nitrificantes

During the oxidation of the substrate, both Nitrosomonas and Nitrobacter have part of the energy made available as high energy phosphate, mamely ADP and ATP. This chemical energy is used to fix CO2. The nature of the reducing power is unknown at present. Active cells of Nitrobacter were shown to fix CO2 along the same pathway as found in higher plant photosynthesis. Sonic extracts of Nitrosomonas and Nitrobacter when incubated with NaH14CO3 and cofactors showed two ports of entry of CO2 into organic compounds one being, as expected, the carboxidismutase reaction. On protein basis an equivalent amount of CO2 was, however, incorporated via the oxaloacetic carboxylase reation. It is clear then that both micoorganisms possess typical autotrophic and heterotrophic mechanisms for the fixation of CO2 which is required for the primary synthesis of cell material.

Advances in pharmacological studies of silymarin

Silymarin is the flavonoids extracted from the seeds of Silybum marianum (L) Gearth as a mixture of three structural isomers: silybin, silydianin and silychristin, the former being the most active component. Silymarin protects liver cell membrane against hepatotoxic agents and improves liver function in experimental animals and humans. It is generally accepted that silymarin exerts a membrane-stabilizing action preventing or inhibiting membrane peroxidation. The experiments with soybean lipoxygenase showed that the three components of silymarin brought about a concentration-dependent non-competitive inhibition of the lipoxygenase. The experiments also showed an analogous interaction with animal lipoxygenase, thus showing that an inhibition of the peroxidation of the fatty acid in vivo was self-evident. Silybin almost completely suppressed the formation of PG at the highest concentration (0.3 mM) and proved to be an inhibitor of PG synthesis in vitro. In our experiments, silybin at lower dose (65 mg/Kg) decreased liver lipoperoxide content and microsomal lipoperoxidation to 84.5% and 68.55% of those of the scalded control rats respectively, and prevented the decrease of liver microsomal cytochrome p-450 content and p-nitroanisole-0-demethylase activity 24 h post-scalding. Effects of silymarin on cardiovascular systen have been studied in this university since 1980. O. O silymarin 800 mg/Kg/d or silybin 600 mg/Kg/d reduced plasma total cholesterol, LDL-C and VLDL-C. They however, enhanced HDL-C in hyperlipenic rats. Further studies showed that silymarin enhanced HDL-C in hyperlipemic rats. Further studies showed that silymarin enhanced HDL-C but didn't affect HDL-C, a property of this component which is beneficial to treatment of atherosclerosis. The results showed silymarin 80 mg or silybin 60 mg decreased in vitro platelet aggregation (porcentagem) in rats. The maximal platelet aggregation induced by ADP declined significantly, and time to reach maximal platelet aggregation and five-minute disaggregation didn't change. In our experiments, iv silybin 22,4 mg/kg lowered the amplitude and duration of diastolic blood pressure (DBP) more than those of systolic (SBP), but the descending aortic blood flow, cardiac contractility and ECG did not change significantly in anesthetized open-chest cats. The results indicated a reduction of peripheral resistance and dilatatory action on the resistant blood vessels. These effects are beneficial to coronary heart disease. We also observed the effects of silybin on morphological change, the release of glutamic oxaloacetate aminotrasferase (GOT) and lactate dehydrogenase (LDH) as well as the radioactivity of 3H-TdR incorporated into DNA in normal cardiac cells and cells infected by coxsackie B5, virus os newborn rats. The results showed that silynin did not affect the morphology of normal cell, and that the pathological change of cells infected by virus was delayed and reduced as compared to control. We have investigated the effect of silybin on synthesis and release of LTs in the cultured porcine cerebral basilar arteries (PCBA). Silybin 100 and 500 µmol/L declined the amounts of LTs released from the PCBA incubsated in the presence of A 23187, AA and indomenthacin. The result suggests that silybin can inhibit the activity of 5-lipoxygenase of cerebral blood vessel and may protect the brain from ischemia.

Use of glass beads and CF 11 cellulose for removal of leukocytes from malaria-infected human blood in field settings

Passage of malaria infected blood through a two-layered column composed of acid-washed glass beads and CF 11 cellulose removes white cells from parasitized blood. However, because use of glass beads and CF 11 cellulose requires filtration of infected blood separately through these two resins and the addition of ADP, the procedure is time-consuming and may be inapropriate for use in the field, especially when large numbers of blood samples are to be treated. Our modification of this process yields parasitized cells free of contaminating leukocytes, and because of its operational simplicity, large numbers of blood samples can be processed. Our procedure also compares well with those using expensive commercial Sepacell resins in its ability to separate leukocytes from whole blood. As a test of usefulness in molecular biologic investigations, the parasites obtained from the blood of malaria-infected patients using the modified procedure yield genomic DNA whose single copy gene, the circumsporozite gene, efficiently amplifies by polymerase chain reaction.

Biomphalaria alexandrina snails as immunogens against Schistosoma mansoni infection in mice

Despite effective chemotherapy, schistosomiasis remains the second largest public health problem in the developing world. Currently, vaccination is the new strategy for schistosomiasis control. The presence of common antigenic fractions between Schistosoma mansoni and its intermediate host provides a source for the preparation of a proper vaccine. The objective of this paper is to evaluate the nucleoprotein extracted from either susceptible or resistant snails to protect against schistosomiasis. The vaccination schedule consisted of a subcutaneous injection of 50 µg protein of each antigen followed by another inoculation 15 days later. Analyses of marker enzymes for different cell organelles [succinate dehydrogenase, lactate dehydrogenase (LDH), glucose-6-phosphatase, acid phosphatase and 5'-nucleotidase] were carried out. Energetic parameters (ATP, ADP, AMP, phosphate potentials, inorganic phosphate, amino acids and LDH isoenzymes) were also investigated. The work was extended to record worm and ova counts, oogram determination in the liver and intestine and the histopathological pattern of the liver. The nucleoprotein of susceptible snails showed reduction in worm and ova counts by 70.96% and 51.31%, respectively, whereas the nucleoprotein of resistant snails showed reductions of 9.67% and 16.77%, respectively. In conclusion, we found that the nucleoprotein of susceptible snails was more effective in protecting against schistosomiasis.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.