Expressão imuno-histoquímica da e-caderina e da ß-catenina em carcinoma epidermóide oral com e sem metástase nodal

The adhesion molecules E-cadherin and β-catenin have been studied as possible markers to distinguish carcinomas with and without metastatic potential. The objective of this research was to study the imunohistochemistry expression of the E-cadherin and β-catenin in oral squamous cell carcinoma (OSCC), aiming to contribute for the better understanding of the biological behavior of this lesion. The sample consisted of 30 cases of OSCC, being 15 of tongue and 15 of lower lip. The profile and intensity of labeling and semi quantitative analysis of the percentage of immunopositive tumoral cells in membrane for E-cadherin and β-catenin had been related with the anatomical localization of the lesion, the presence or not of nodal metastasis and the histological grade of malignancy in the invasive front area of the tumor. It was registered the presence or not of cytoplasmic and nuclear labeling of the β-catenin. The results had been submitted to the statistical analysis, being used the Mann-Whitney Test, the Fisher Test and the Spearman Correlation Coefficient (α=0, 05). The results showed that the expression in membrane for E-cadherin and β-catenin was, predominantly, the heterogeneous profile in the lower lip and tongue carcinomas, as well as in the cases with and without nodal metastasis. It was not observed significant statistical difference between expression profile and amount of immunopositive cells for E-cadherin, β-catenin and the anatomical localization of the lesion and for the presence or not of nodal metastasis. However, there was significant difference of the reduced expression of these proteins with the high score of malignancy. It was verified that the expression of the β-catenin in cytoplasm was present in 22 (73.33%) of the 30 analyzed cases, and 6 cases (20%) showed nuclear expression. The statistical analysis detected significant association between the expression of the β-catenin in the cytoplasm with the histological grade of malignancy, being this molecule more frequently present in the cytoplasm in the cases of high score of malignancy. It was concluded that the reduced immunoexpression of these proteins in membrane can be related with the lowest cellular differentiation, as well as with the pattern of invasion in nests and isolated cells, demonstrated in the cases of OSCC with high histological grade of malignancy

Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Atividade bioinseticida e mecanismo de ação de vicilinas de sementes Erythrina velutina sobre moscas-das frutas Ceratitis capitata

The fruit fly Ceratitis capitata is considered the most destructive pest of the world fruitculture. Many pest management practices, mainly based on agrochemicals, have been developed to allow the world-wide commerce of fruit. Solutions to decrease the use of synthetic insecticides in agriculture are based on the development of new target-specific compounds which cause less damage to the environment, especially vegetal proteins with insecticidal effects. The aim of this work was to evaluate the deleterious effect of a purified vicilin of E. velutina (EvV) seeds to C. capitata larvae and adult insects and to investigate the mechanisms involved in these effects. EvV was purified, characterized and its deleterious effect was tested in bioassay systems. EvV mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV is a glycoprotein with affinity to chitin. Its molecular weight, of 216,57 kDa, was determined by gel filtration chromatography in FPLC system. Using SDS-PAGE, it was possible to observe EvV dissociation in two main subunits of 54,8 and 50,8 kDa. When it was submitted to eletrophoresis in native conditions, EvV presented only one band of acid characteristic. The WD50 and LD50 values found in the bioassays were 0,13% and 0,14% (w/w), respectively for the larvae. EvV deleterious effects were related to the binding to chitin structures presented in peritrophic membrane and gut epithelial cells, associated with its low digestibility in C. capitata digestive tract. The results described herein are the first demonstration of the larvicidal effects of plant protein on C. capitata larvae. EvV may be part of the pest management programs, in the toxic bait composition, or an alternative in plant improvement program

Determinação de motivos de ligação à quitina em vicilinas de Canavalia ensiformis e Vigna unguiculata através de métodos in silico e relação com suas toxicidades para o bruquídeo Callosobruchus maculatus (Coleoptera:Bruchidae)

Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Investigação do papel modulador do Haloperidol sobre os níveis de proteínas relacionadas à plasticidade e preferência por objetos novos em camudongos

Dopamine (DA) is known to regulate both sleep and memory formations, while sleep plays a critical role in the consolidation of different types of memories. We believe that pharmacological manipulation of dopaminergic pathways might disrupt the sleep-wake cycle, leading to mnemonic deficits, which can be observed in both behavioral and molecular levels. Therefore, here we investigated how systemic injections of haloperidol (0.3 mg/kg), immediately after training in dark and light periods, affects learning assessed in the novel object preference test (NOPT) in mice. We also investigated the hippocampal levels of the plasticity-related proteins Zif-268, brain-derived neurotrophic factor (BDNF) and phosphorylated Ca2+/calmodulin-dependent protein kinases II (CaMKII-P) in non-exposed (naïve), vehicle-injected controls and haloperidol-treated mice at 3, 6 and 12 hours after training in the light period. Haloperidol administration during the light period led to a subsequent impairment in the NOPT. In contrast, preference was not observed during the dark period neither in mice injected with haloperidol, nor in vehicle-injected animals. A partial increase of CaMKII-P in the hippocampal field CA3 of vehicle-injected mice was detected at 3h. Haloperidol-treated mice showed a significant decrease in the dentate gyrus of CaMKII-P levels at 3, 6 and 12h; of Zif-268 levels at 6h, and of BDNF levels at 12h after training. Since the mnemonic effects of haloperidol were only observed in the light period when animals tend to sleep, we suggest that these effects are related to REM sleep disruption after haloperidol injection

Membrana de alumina anódica: comportamento da microestrutura e estudo das propriedades ópticas após tratamento térmico

Thin commercial aluminum electrolytic and passed through reactions was obtained with anodic alumina membranes nanopores. These materials have applications in areas recognized electronic, biomedical, chemical and biological weapons, especially in obtaining nanostructures using these membranes as a substrate or template for processing nanowires, nanodots and nanofibers for applications noble. Previous studies showed that the membranes that have undergone heat treatment temperature to 1300° C underwent changes in morphology, crystal structure and optical properties. This aim, this thesis, a study of the heat treatment of porous anodic alumina membranes, in order to obtain and to characterize the behavior changes structures during the crystallization process of the membranes, at temperatures ranging between 300 and 1700° C. It was therefore necessary to mount a system formed by a tubular furnace resistive alumina tube and controlled environment, applying flux with special blend of Ag-87% and 13% N2, in which argon had the role of carrying out the oxygen nitrogen system and induce the closing of the pores during the densification of the membrane. The duration of heat treatment ranged from 60 to 15 minutes, at temperatures from 300 to 1700° C respectively. With the heat treatment occurred: a drastic reduction of porosity, grain growth and increased translucency of the membrane. For the characterization of the membranes were analyzed properties: Physical - thermogravimetric, X-ray diffraction, BET surface area; morphological - SEM, EDS through compositional and, optical absorbance, and transmittance in the UV-VIS, and FTIR. The results using the SEM showed that crystallization has occurred, densification and significant changes in membrane structure, as well as obtaining microtube, the BET analysis showed a decrease in specific surface area of the membranes has to 44.381 m2.g-1 to less than 1.8 m2.g-1 and in the analysis of transmittance and absorbance was found a value of 16.5% in the range of 800 nm, characteristic of the near infrared and FTIR have confirmed the molecular groups of the material. Thus, one can say that the membranes were mixed characteristics and properties which qualify for use in gas filtration system, as well as applications in the range of optical wavelength of the infra-red, and as a substrate of nanomaterials. This requires the continuation and deepening of additional study

Biodegradação de hidrocarbonetos aromáticos policíclicos: prospecção metagenômica e modelagem computacional 3-D de proteínas

Knowledge of the native prokaryotes in hazardous locations favors the application of biotechnology for bioremediation. Independent strategies for cultivation and metagenomics contribute to further microbiological knowledge, enabling studies with non-cultivable about the "native microbiological status and its potential role in bioremediation, for example, of polycyclic aromatic hydrocarbons (HPA's). Considering the biome mangrove interface fragile and critical bordering the ocean, this study characterizes the native microbiota mangrove potential biodegradability of HPA's using a biomarker for molecular detection and assessment of bacterial diversity by PCR in areas under the influence of oil companies in the Basin Petroleum Geology Potiguar (BPP). We chose PcaF, a metabolic enzyme, to be the molecular biomarker in a PCR-DGGE detection of prokaryotes that degrade HPA s. The PCR-DGGE fingerprints obtained from Paracuru-CE, Fortim-CE and Areia Branca-RN samples revealed the occurrence of fluctuations of microbial communities according to the sampling periods and in response to the impact of oil. In the analysis of microbial communities interference of the oil industry, in Areia Branca-RN and Paracuru-CE was observed that oil is a determinant of microbial diversity. Fortim-CE probably has no direct influence with the oil activity. In order to obtain data for better understanding the transport and biodegradation of HPA's, there were conducted in silico studies with modeling and simulation from obtaining 3-D models of proteins involved in the degradation of phenanthrene in the transport of HPA's and also getting the 3-D model of the enzyme PcaF used as molecular marker in this study. Were realized docking studies with substrates and products to a better understanding about the transport mechanism and catalysis of HPA s

Proteínas SAG1, SAG2, SAG3 de toxoplasma gondii como perspectivas para o desenvolvimento de protótipo vacinal contra a toxoplasmose

Toxoplasmosis is a zoonosis of worldwide distribution caused by the protozoan Toxoplasma gondii, triggering dangerous complications in immunocompromised patients and pregnant women, as well as having great economic impact for the livestock. So far the control of toxoplasmosis is made primarily by chemotherapy. However, most drugs used routinely have some limitations. In order to control this disease, several research groups, including ours, has been working to develop a medical-veterinary vaccine based on parasite antigens, vectors and protocols of immunization. In this study were implemented and standardized methodologies for amplification and cloning of recombinant immunogens in the system for the development of a prototype vaccine, based on the surface antigens of T. gondii and recombinant adenovirus encoding these antigens. Genes encoding BAG1, GRA2 and SAG1 proteins were amplified. We established a strategy for cloning SAG1, SAG2, SAG3 and TgAMA1- genes in recombinant system. The genes encoding SAG1 and SAG2 were cloned and their sequences showed high similarity with sequences from GenBank. The virtual translation of these proteins showed polymorphisms in the amino acid sequence, which can be correlated with levels of antigenicity. Simultaneously, the adenovirus encoding the SAGs (HAdSAGs) were expanded, purificated and characterizated. Immunization of C57bl/6 mice, using viral supernatant was not enought to elicit immune responses at high levels, being required HAdSAGs titration for future immunizations. Therefore, this study allowed the cloning of the two genes important for the development of a prototype vaccine. Besides, implementations methodologies that permit advancements in the development of a vaccine against toxoplasmosis using adenovirus to express proteins of the parasite

Comparação de efeitos dos extratos de Hypericum perforatum (Hipérico) e de Mentha crispa (Hortelã) em diferentes modelos experiemtais

Several clinic evaluations have been possible with radiobiocomplexes labeled with technetium-99m (99mTc). Some natural and synthetic drugs are capable of to interfere on the labeling of blood constituents with 99mTc, as well as on the biodistribution of radiobiocomplexes. Authors have also reported about the toxicity of several natural products. The aim of this study was to compare the effects of the Mentha crispa (hortelã) and of the Hypericum perforatum (hipérico) in different experimental models. On the labeling of red blood cells (RBC) and plasma and cellular proteins with 99mTc, both extracts were capable of to decrease the radioactivity percentage on the cellular compartment and on the fixation on plasma and cellular proteins. On the morphometry of the RBC, only the hortelã was capable to alter the shape and the perimeter/area ratio of the RBC. On the biodistribution of the radiobiocomplex sodium pertechnetate (Na99mTcO4), the hortelã increased the Na99mTcO4 distribution in the kidney, spleen, liver and thyroid, meanwhile the hipérico decreased the Na99mTcO4 distribution in the bone, stomach, lungs and thyroid, and increased the Na99mTcO4 distribution in the pancreas. On the bacterial cultures survival, the hipérico was capable of to protect the bacteria against the stannous chloride (SnCl2) effect. The hipérico did not alter the topology of plasmidial DNA and did not protect the plasmidial DNA against the SnCl2 action. Probably, the effects presented by both extracts could be due to chemical compounds of the extracts that could alter the morphology of the RBC and the plasma membrane ions transport, and/or by phytocomplexes that could be formed with different effects dependent on the biological system considered

Avaliação de efeitos de um extrato aquoso de cinnamomum zeylanicum L. na marcação de constituintes sanguíneos com tecnécio-99m e na morfologia das hemácias de ratos wistar

Radiobiocomplexes are used to obtain images in nuclear medicine and employed in basic research. Blood constituents labeled with technetium-99m (99mTc) have also been employed as radiobiocomplexes and used also experimental model for evaluation of the biological effects of natural or synthetic drugs. The analysis of the morphology and the morphometrics parameters (perimeter/área ratio) can be used to evaluate the effects of drugs upon the structure of the membrane of red blood cells. Cinnamomum zeylanicum (cinnamon) is a spice used as herbal medicine to treat diseases. The aim of this study was to evaluate the effect of in vitro and in vivo treatment with an aqueous cinnamon extract on the labeling of blood constituents with 99mTc and on the morphology of red blood cells from Wistar rats. In the in vitro treatment, isolated blood sample from animals were incubated with cinnamon extract. In the in vivo treatment, blood samples were also withdrawn from animals treated with cinnamon extract. In both cases, the radiolabeling of blood constituents was done. The morphological analysis of red blood cells was also done. As control, blood or animals treated with NaCl 0.9%. The data obtained on the labeling of blood constituents with 99mTc experiments indicated that the in vitro treatment with cinnamon extract was capable to decrease signiicantly (p<0.05) the percentage of radioactivity in cellular compartments and on the fixation of cellular and plasma proteins. These effects were not observed on the in vivo treatment. The results obtained for the morphology of red blood cells suggest that the in vitro and in vivo treatments did not alter the morphology and the perimeter/area ratio. The in vitro treatment with aqueous cinnamon extract could affect the membrane structures related with the oxidation status of the stannous ion pertechnetate ion, altering the labeling of blood constituentes with 99mTc. This study was a multidisciplinary experimental research. It was developed with the contribution of the different Departments and Services of the Hospital Universitário Pedro Ernesto of the Universidade do Estado do Rio de Janeiro

Efeito de um extrato de Artemísia Vulgaris L. marcação in vitro de constituintes sanguíneos com tecnécio-99m em ratos wistar

Artemisia vulgaris L..is used in folk medicine and in Traditional Chinese Medicine (TCM). This medicinal plant has been utilized as anticonvulsive, analgesic, antispasmodic effect, rheumatic pains, menstrual dyspepsia, asthenia, epilepsy, hepatitis, fevers, anemia and to expel parasites. In nuclear medicine, blood constituents are labeled with technetium-99m (99mTc) and used as radiopharmaceuticals (radiobiocomplexes). Authors have been described that synthetic and/or natural drugs could modify the labeling of blood constituents with 99mTc. The aim of this work was to evaluate the effects of an aqueous extract of Artemisia vulgaris L. on the labeling of blood constituents with 99mTc. Blood samples withdrawn of Wistar rats were incubated with Artemisia vulgaris L, stannous chloride and 99mTc, as pertechnetate ion. Aliquots of plasma (P) and blood cells (BC) were isolated. Aliquots of P and BC were also precipitated with trichloroacetic acid and soluble (SF) and insoluble (IF) fractions were separated. The radioactivity in each fraction was counted and the percentages of radioactivity (%ATI) were calculated. Artemisia vulgaris L. extract decreased significantly (p<0.05) the %ATI on BC and on IF-BC. The analysis of the results indicates that the extract could have substances that could interfere on the transport of stannous through the erythrocyte membrane altering the labeling of blood cells with 99mTc. Working in this study was a multidisciplinary group, with Phisical therapists, Biomedicals, Physicals, Pharmacists, Biologists, Statistics and Physicians.

Extração, caracterização e atividades biológicas de proteínas da espécie cnidoscolus urens (L.) Arthur

The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocaseína and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The Aα chain and Bβ of fibrinogen were completely cleaved at a concentration of 0.18 μg/μL of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60ºC. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory