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Introduction: Diplotropis incexis is popularly known as sucupira. To extract its oil, which is used for the treatment of eczema, inflammation, skin patches, rheumatism, and to treat hemorrhages, it is necessary to breakdown and decoct the seeds. The aim was to evaluate the antioxidant activity and cytotoxic effect of Diplotropis incexis oil (DIO) in B16-F10 murine melanoma cells. Materials and methods: DIO was obtained from the breakdown of seeds, Soxhlet extraction, and rotary evaporation. Antioxidant activity was measured by radical scavenging. Quantification of phenolic groups was performed using a standard curve of gallic acid and quercetin. DIO toxicity was determined between 0.01 – 100 µg/mL by photometry (MTT) and cytometry. Finally, cell morphology was examined by confocal microscopy. Results: The DIO showed antioxidant potential, being about 335x less potent than the gallic acid control. 109 mg of total phenols and 381 mg of flavonoids were found for every 100g of crude oil. The DIO showed high cytotoxicity against the B16-F10 line, presenting IC50 = 10 µg/mL for cells treated for 48 hours. The cells underwent drastic morphological changes, presenting changes in membrane structure and drastic changes in the cytoskeleton, due to F-actin filament disarrangement. Conclusions: The first reports of DIO activity showed that it has moderate antioxidant activity with the presence of phenolic groups in its composition. In addition to having cytotoxic activity in the B16-F10 cell lines, it is able to promote decreased cell viability and morphological changes depending on concentration and time spent in contact with oil.

Lantana camara L. (Verbenaceae) e uma planta de caule lenhoso, ramos eretos e com inflorescencia com bractea-involucrais externas, flor com calice menor que 2.5/4.5 lobado, corola amarela/vermelha/laranja maior que as bracteas e o fruto e da cor vinacea com mesocarpo do tipo carnoso de ocorrencia mundial. Esta especie se adapta facilmente em regioes com acao antropica. Na familia, L. camara e uma das mais estudadas no mundo como planta medicinal, entretanto, raros sao os estudos histoquimicos envolvendo partes vegetativas desta especie. O estudo teve como objetivo identificar e localizar in situ metabolitos presentes nas folhas de L. camara ocorrente em area de Mata Atlântica, no sul da Bahia, Brasil. Cortes realizados a mao livre em folhas frescas e fixadas em formalina neutra tamponada de especimes coletados na localidade de Marau, Bahia, Brasil foram submetidas a histoquimica e observacao microscopica. Alcaloides foram identificados em tricomas glandulares e nos idioblastos; lipideos em tricomas glandulares, no parenquima palicadico e na cuticula; lignina, nas celulas do xilema; compostos fenolicos no parenquima palicadico, nos tricomas glandulares e oleos essenciais nas celulas do xilema, do floema e no feixe vascular. Os resultados deste trabalho corroboraram estudos anteriores e agregaram conhecimento com a descricao da presenca de alcaloides, ainda nao descritos nas folhas de L. camara por metodo histoquimico que possibilitarao direcionamento para pesquisas futuras sobre o uso da planta.

PURPOSE: There is a growing scientific interest in the plasticity and therapeutic potential of adipose-derived stem cells (ASCs), which are multipotent and abundant in adipose tissue and can differentiate in vitro into multiple lineages, including adipocytes, chondrocytes, osteoblasts, neural cells, endothelial cells and cardiomyocytes. The aim of this study was to isolate, cultivate and identify ASCs. METHODS: Human adipose precursor cells were obtained from subcutaneous abdominal tissue. Recently dispersed cells were separated by density centrifugation gradient, cultured and then analyzed. RESULTS: Human ASCs were able to replicate in our culture conditions. The cells maintained their phenotypes throughout the studied period on different passages confirming they suitability for in vitro cultivation. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. Flow cytometry results showed that these cells expressed CD73, CD90 and CD105, (mesenchymal stem-cells markers), contrasting with the lack of expression of CD16, CD34 and CD45 (hematopoietic cells markers). CONCLUSION: It was possible to isolate human adipose-derived stem cells by in vitro cultivation without adipogenic induction, maintaining their functional integrity and high proliferation levels. The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. OBJETIVO: Há um interesse científico crescente na plasticidade e potencial terapêutico das células-tronco do tecido adiposo humano, células multipotentes e abundantes no tecido adiposo que podem se diferenciar in vitro em múltiplas linhagens celulares, incluindo adipócitos, condrócitos, osteoblastos, células neurais, endoteliais e cardiomiócitos. O objetivo deste estudo foi isolar, cultivar e identificar células-tronco do tecido adiposo humano. MÉTODOS: Células precursoras humanas do tecido adiposo foram obtidas de tecido abdominal subcutâneo. As células recém-dispersas foram separadas por gradiente de centrifugação por densidade, cultivadas e então analisadas. RESULTADOS: As células-tronco do tecido adiposo humano foram capazes de se replicar nas nossas condições de cultivo e mantiveram seu fenótipo em diferentes passagens durante o estudo, confirmando sua adequabilidade para cultivo in vitro. A diferenciação adipogênica, osteogênica e condrogênica também foi induzida, confirmando seu potencial de células-tronco mesenquimais in vitro. Os resultados de citometria de fluxo evidenciaram a expressão dos marcadores de células-tronco mesenquimais CD73, CD90 e CD105, contrastando com a falta de expressão dos marcadores de células hematopoiéticas CD16, CD34 e CD45. CONCLUSÃO: Foi possível isolar células-tronco do tecido adiposo humano por cultivo in vitro sem indução adipogênica, mantendo sua integridade funcional e altos níveis de proliferação. As células demonstraram potencial de diferenciação adipogênico, osteogênico e condrogênico in vitro.

O extrato do Ginkgo biloba (Egb 761) é conhecido por sua capacidade de eliminar as espécies reativas de oxigênio (EROs). Estudos demonstram que sua propriedade antioxidante pode ser utilizada tanto na prevenção quanto na terapia de doenças associadas ao estresse oxidativo. Além do efeito antioxidante e inibidor da peroxidação lipídica, o Ginkgo biloba está envolvido na apoptose de células tumorais. Seu extrato é capaz de inibir a angiogênese por meio da regulação negativa do gene VEGF. Esses dados o apontam como um potente agente antitumoral com bons prospectos na aplicação clínica.

OBJETIVOS: Avaliar a morfologia das organelas e do citoesqueleto em células pancreáticas humanas cultivadas e a mobilização de Ca2+ em resposta à glicose e ACh por medidas fluorimétricas. MATERIAL E MÉTODOS: As células foram semeadas em lamínulas, fixadas e marcadas com uma combinação de fluoróforos: o núcleo foi corado com DAPI e as mitocôndrias, com Mytotracker Red. Foram utilizados faloidina e anticorpos secundários conjugados com Alexa Fluor verde e vermelho fluorescentes (488 e 594) para identificar proteína actina F e receptor muscarínico tipo M3, respectivamente. Para estudar a mobilização de Ca2+, as células foram incubadas com fura-2/AM. RESULTADOS: As células pancreáticas humanas apresentaram morfologia preservada com grande quantidade de mitocôndrias. Na região de maior densidade celular, evidenciou-se as pseudo-ilhotas e os receptores muscarínicos M3. Por meio da elevação da [Ca2+]c, devido à ação da glicose e ACh, mostrou-se preservação da capacidade responsiva a esses estímulos e foi dependente de concentração desses agonistas. A glicose promoveu uma resposta sustentada e a ACh induziu uma resposta bifásica. CONCLUSÃO: As células pancreáticas humanas cultivadas conservaram sua morfologia. A mobilização de Ca2+ em resposta à glicose e a ACh confirma a sua funcionalidade. Os receptores muscarínicos M3 estão presentes nessas células. AIMS: The proposal of this study was to analyze morphology of the organelles and cytoskeleton in human pancreatic cells cultured and the mobilization of the cytosolic calcium ([Ca2+]c) in response to glucose and ACh by fluorimetry method. MATERIAL AND METHODS: The cells were plated on glass coverslips, fixed and stained with a combination of fluorophores: the nuclei were stained with DAPI and mitochondria with Mytotracker Red. It was used phalloidin and the secondary antibodies Alexa Fluor conjugated green and red-fluorescent (488 and 594) to identify the protein cell actin F and type M3 muscarinic receptor respectively. The cells also were loaded with fura-2/AM to study Ca2+ mobilization. RESULTS: The human pancreatic cells show characteristics morphologically preserved with great amount of mitochondria. In region major cell density was evidenced pseudo-islets and type M3 muscarinic receptors. Through increase of [Ca2+]c due to action of glucose and ACh were shown that the cells’ capacity to respond to these stimuli were conserved. The elevation of the [Ca2+]c depended on concentration by glucose-induced promoting sustained phase and ACh-induced a biphasic response. CONCLUSION: The morphologic characteristics of human pancreatic cells cultured were preserved. The Ca2+ mobilization in response to glucose and ACh confirmed its functionality. The expression of the M3 muscarinic receptors in human pancreatic cell cultured was demonstrated.

We have previously demonstrated that Chinese hamster ovary (CHO) cells transfected with the angiotensin II AT 1 receptor gene containing only the coding region, presented tachyphylaxis to the total inositol phosphate (InsP s ) and Ca 2+ responses mediated by angiotensin II and [2-lysine]angiotensin II ([Lys 2 ]angiotensin II). Now we have evaluated the possible role of the 3′-untranslated region of the angiotensin AT 1 receptor mRNA in modulating the angiotensin AT 1 receptor-mediated cellular responses. The binding parameters, as well as the Ca 2+ and InsP s responses induced by angiotensin II and [Lys 2 ]angiotensin II were similar in cells transfected with the angiotensin AT 1 receptor with or without the 3′-untranslated region sequence. In cells transfected with the receptor containing the 3′-untranslated region sequence, angiotensin II-induced Ca 2+ and InsP s responses were desensitized by repeated stimulations, whereas [Lys 2 ]angiotensin II caused desensitization of InsP s production but not of Ca 2+ uptake in these cells. Our results suggest that the 3′-untranslated region plays a role in modulating cell signalling involved in the tachyphylaxis of angiotensin AT 1 receptor-mediated Ca 2+ responses.

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1receptor were designed to explore a possible role of a leucine at two positions, Leu265and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.Key words: angiotensin II, AT1receptor, site-directed mutagenesis.

The hematopoietic system is organized in a hierarchical manner in which rare hematopoietic stem cells initiate the hierarchy and have the ability to self-renew, proliferate and differentiate into different lineages of peripheral blood cells as well as to intermediate hematopoietic progenitor cells. Most hematopoietic stem cells are quiescent under steadystate conditions and function as a stock population to protect the hematopoietic system from exhaustion due to various stressful conditions. In contrast, hematopoietic progenitor cells are rapidly proliferating cells with limited self-renewal ability. The proliferation and differentiation of hematopoietic progenitor cells fulfills the requirements of normal hematopoiesis allowing the hematopoietic system to react promptly and effectively to meet the demand for increasing the output of mature cells during hematopoietic crisis such as loss of blood, hemolysis, infection, the depletion of HPCs by chemotherapy and/or radiotherapy (Reya, 2003; Weissman et al., 2001; Walkley et al., 2005).

Cytochrome c-mediated apoptosis in cells submitted to photodynamic therapy raises the question about the ability of photodynamically oxidized cytochrome c ( cytc405) to trigger apoptosis as well as the effect of membranes on protein photo-oxidation. Cytochrome c was submitted to irradiation in the presence of MB+ in phosphate buffer and in the presence of four types of phosphatidylcholine/phosphatidylethanolamine/cardiolipin ( PCPECL) liposomes ( 50/30/20%): totally saturated lipids ( tsPCPECL), totally unsaturated lipids ( tuPCPECL), partially unsaturated ( 80%) lipids, with unsaturation in the PC and PE content ( puPCPECL80), and partially unsaturated ( 20%) lipids, with unsaturation in the CL content ( puPCPECL20). Cytc405 was formed by irradiation in buffered water and in tsPCPECL and puPCPECL20 liposomes. in the presence of tuPCPECL and puPCPECL80, cytochrome c was protected from photodynamic damage ( lipid-protected cytochrome c). in CL liposomes, 25% unsaturated lipids were enough to protect cytochrome c. the presence of unsaturated lipids, in amounts varying according to the liposome composition, are crucial to protect cytochrome c. Interesting findings corroborating the unsaturated lipids as cytochrome c protectors were obtained from the analysis of the lipid-oxidized derivatives of the samples. Native cytochrome c, lipid-protected cytochrome c, and cytc405 were microinjected in aortic smooth muscle cells. Apoptosis, characterized by nucleus blebbing and chromatin condensation, was detected in cells loaded with native and lipid protected cytochrome c but not in cells loaded with cytc405. These results suggest that photodynamic therapy-promoted apoptosis is feasible due to the protective effect of the mitochondrial lipids on the cytochrome c structure and function. Univ Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, BR-08780911 São Paulo, Brazil Universidade Federal de São Paulo, Dept Farmacol, São Paulo, Brazil Universidade Federal de São Paulo, Dept Farmacol, São Paulo, Brazil Web of Science

The proposal of this study was to analyze morphology of the organelles and cytoskeleton in human pancreatic cells cultured and the mobilization of the cytosolic calcium ([Ca2+]c) in response to glucose and ACh by fluorimetry method.The cells were plated on glass coverslips, fixed and stained with a combination of fluorophores: the nuclei were stained with DAPI and mitochondria with Mytotracker Red. It was used phalloidin and the secondary antibodies Alexa Fluor conjugated green and red-fluorescent (488 and 594) to identify the protein cell actin F and type M3 muscarinic receptor respectively. The cells also were loaded with fura-2/AM to study Ca2+ mobilization.The human pancreatic cells show characteristics morphologically preserved with great amount of mitochondria. In region major cell density was evidenced pseudo-islets and type M3 muscarinic receptors. Through increase of [Ca2+]c due to action of glucose and ACh were shown that the cells capacity to respond to these stimuli were conserved. The elevation of the [Ca2+]c depended on concentration by glucose-induced promoting sustained phase and ACh-induced a biphasic response.The morphologic characteristics of human pancreatic cells cultured were preserved. The Ca2+ mobilization in response to glucose and ACh confirmed its functionality. The expression of the M3 muscarinic receptors in human pancreatic cell cultured was demonstrated.

The manifestation of tachyphylaxis to angiotensin II in Chinese hamster ovary (CHO) cells expressing the rat angiotensin II AT(1) receptor was investigated. The cells were transfected with a cDNA fragment containing the complete coding region of the angiotensin II AT(1A) receptor gene, as well as 56 bp of its 3'- and 52 bp of its 5'-untranslated regions. These cells (CHO-AT(1)) responded to angiotensin II by increases in intracellular Ca(2+) concentration and inositol phosphate turnover, which were inhibited upon repeated administrations, characterizing the tachyphylaxis phenomenon. In contrast to smooth muscle cells, which are rendered tachyphylactic to angiotensin II but not to [2-lysine]angiotensin II ([Lys(2)]angiotensin II), this analogue induced responses in CHO-AT(1) cells that were also inhibited upon repeated administrations. A smooth muscle cell line, which showed tachyphylaxis only to angiotensin II, became tachyphylactic also to [Lys(2)]angiotensin II after transfection with the angiotensin II AT(1) receptor gene. Our findings suggest that posttranscriptional control directed by the 3'- or the 5'-untranslated regions in the angiotensin II AT(1) receptor gene may play a role in modulating the signal transduction pathways involved in the mechanism of angiotensin II tachyphylaxis.