Antineoplasic activity of Copaifera multijuga oil and fractions against ascitic and solid Ehrlich tumor

The aim of this study was to investigate the effect of chronic treatment with C. multijuga oil on Ehrlich tumor evolution. C multijuga was fractionated in a KOH impregnated silica gel column chromatography to give three distinct fractions, i.e., hexanic, chloroformic, and methanolic, mainly composed by hydrocarbon sesquiterpenes, oxygenated sesquiterpenes and acidic diterpenes, respectively. Results demonstrated that the C multijuga oil, the hexanic, and chloroformic fractions did not develop toxic effects. The oil, hexanic and chloroformic fractions (doses varying between 100 and 200 mg/kg) showed antineoplasic properties against Ehrlich ascitic tumor (EAT) and solid tumor during 10 consecutive days of treatment inhibiting ascitic tumor cell number, reverting medulla and blood cell counts to values similar to control group, and inhibiting the increase on several inflammatory mediators (total protein, PGE(2), nitric oxide, and TNF) on ascitic fluid. The treatment also inhibited the increase in paw volume on tumor-inoculated mice. In conclusion, C. multijugo as well as its fractions demonstrated antineoplasic effect even after oral administration confirming its use by traditional medicine. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Produção in vitro, análise fitoquímica e avaliação da atividade antineoplásica de metabólitos de Hovenia dulcis Thunb. (Rhamnaceae) obtidos por diferentes estratégias biotecnológicas

Hovenia dulcis Thunberg, natural da Ásia Oriental, é cultivada no Brasil onde é conhecida como uva-do-japão. A espécie possui várias indicações na medicina popular e alguns estudos apontam o seu potencial antineoplásico, tripanocida e hepatoprotetor. Metabólitos secundários são substâncias não essenciais para a sobrevivência celular, mas que fornecem vantagens adaptativas aos vegetais, sendo atribuído, para algumas delas, atividades biológicas importantes. Substâncias de interesse medicinal têm sido obtidas por técnicas da cultura de tecidos vegetais, como a calogênese e a cultura de células em suspensão, que permitem a síntese de matéria-prima de forma contínua e homogênea, independentemente de fatores ambientais e sazonais. O presente estudo objetivou o estabelecimento de culturas in vitro de H. dulcis, visando à produção de metabólitos de interesse, com vistas à avaliação do seu potencial antineoplásico sobre células K562. Foram testados protocolos para o estabelecimento de diferentes sistemas, como culturas de calos, de células em suspensão (CCS) e compact callus clusters (CCC) e ainda a avaliação do uso de elicitores na otimização de metabólitos produzidos in vitro. Foi verificado que a adição dos fitorreguladores KIN e TDZ, substituindo o BAP, não foi capaz de induzir a formação de calos friáveis, bem como a manutenção das culturas em ausência de luz. O uso do nitrato de prata promoveu a friabilidade de calos em todas as concentrações testadas, considerando-se 2,0 mg.L-1 a melhor concentração. Foram alcançadas taxas de 100% de formação de CCS tanto na presença, quanto em ausência de AgNO3. O maior acúmulo de biomassa foi verificado na concentração mais baixa de PIC (0,625 mg.L-1). A análise dos espectros de RMN indicou a presença de (+)-dihidromiricetina, (+)-galocatequina, hovenitina II, hovenosideo G, hodulosideo III, hodulosideo IV, hodulosideo I e hovenidulciosideo B1 nas culturas de calos friáveis. No estabelecimento de culturas CCC, observou-se a formação de calos compactos verdes em todas as concentrações de ANA testadas. O aumento da velocidade de rotação para 135 rpm aumentou a dispersão das células com consequente formação dos agregados celulares desejados. A seleção de linhagens celulares demonstrou ser um método eficiente na uniformização do tamanho desses agregados e tal uniformidade se manteve estável por mais de cinco subcultivos em 100% das culturas. Uma fração rica em saponinas foi obtida a partir dos agregados celulares, correspondendo a 1,46% da massa seca. A análise por RMN sugeriu a presença das saponinas Hovenosideo G e dos hovenidulciosideos A2 e B2. O uso de elicitores em cultura de calos mostrou-se adequado à produção de metabólitos secundários, sem alterações morfológicas nos mesmos. A elicitação alterou o perfil cromatográfico analisado por HPLC. Na elicitação com 5,0 mg.L-1 de extrato de levedura foi verificado um aumento de quase três vezes (12,280 3,396 equivalentes de quercetina/mg de extrato) na síntese de flavonoides. Finalmente, os estudos de ação antitumoral in vitro demonstraram citotoxicidade dos extratos de calos não elicitados de H. dulcis sobre linhagem de leucemia mieloide crônica (IC50 de 74,05 μg.mL-1.) e inibição do crescimento de tais células (K562), sugerindo o potencial antineoplásico para um produto biotecnológio (calo) desta espécie.

Comparison of the defensive elicitation activity of cerato-populin and cerato-platanin: a structural model

Plants can defend themselves from potential pathogenic microorganisms relying on a complex interplay of signaling pathways: activation of the MAPK cascade, transcription of defense related genes, production of reactive oxygen species, nitric oxide and synthesis of other defensive compounds such as phytoalexins. These events are triggered by the recognition of pathogen’s effectors (effector-triggered immunity) or PAMPs (PAMP-triggered immunity). The Cerato Platanin Family (CPF) members are Cys-rich proteins secreted and localized on fungal cell walls, involved in several aspects of fungal development and pathogen-host interactions. Although more than hundred genes of the CPF have been identified and analyzed, the structural and functional characterization of the expressed proteins has been restricted only to few members of the family. Interestingly, those proteins have been shown to bind chitin with diverse affinity and after foliar treatment they elicit defensive mechanisms in host and non-host plants. This property turns cerato platanins into interesting candidates, worth to be studied to develop new fungal elicitors with applications in sustainable agriculture. This study focus on cerato-platanin (CP), core member of the family and on the orthologous cerato-populin (Pop1). The latter shows an identity of 62% and an overall homology of 73% with respect to CP. Both proteins are able to induce MAPKs phosphorylation, production of reactive oxygen species and nitric oxide, overexpression of defense’s related genes, programmed cell death and synthesis of phytoalexins. CP, however, when compared to Pop1, induces a faster response and, in some cases, a stronger activity on plane leaves. Aim of the present research is to verify if the dissimilarities observed in the defense elicitation activity of these proteins can be associated to their structural and dynamic features. Taking advantage of the available CP NMR structure, Pop1’s 3D one was obtained by homology modeling. Experimental residual dipolar couplings and 1H, 15N, 13C resonance assignments were used to validate the model. Previous works on CPF members, addressed the highly conserved random coil regions (loops b1-b2 and b2-b3) as sufficient and necessary to induce necrosis in plants’ leaves: that region was investigated in both Pop1 and CP. In the two proteins the loops differ, in their primary sequence, for few mutations and an insertion with a consequent diversification of the proteins’ electrostatic surface. A set of 2D and 3D NMR experiments was performed to characterize both the spatial arrangement and the dynamic features of the loops. NOE data revealed a more extended network of interactions between the loops in Pop1 than in CP. In addition, in Pop1 we identified a salt bridge Lys25/Asp52 and a strong hydrophobic interaction between Phe26/Trp53. These structural features were expected not only to affect the loops’ spatial arrangement, but also to reduce the degree of their conformational freedom. Relaxation data and the order parameter S2 indeed highlighted reduced flexibility, in particular for loop b1-b2 of Pop1. In vitro NMR experiments, where Pop1 and CP were titrated with oligosaccharides, supported the hypothesis that the loops structural and dynamic differences may be responsible for the different chitin-binding properties of the two proteins: CP selectively binds tetramers of chitin in a shallow groove on one side of the barrel defined by loops b1-b2, b2-b3 and b4-b5, Pop1, instead, interacts in a non-specific fashion with oligosaccharides. Because the region involved in chitin-binding is also responsible for the defense elicitation activity, possibly being recognized by plant's receptors, it is reasonable to expect that those structural and dynamic modifications may also justify the different extent of defense elicitation. To test that hypothesis, the initial steps of a protocol aimed to the identify a receptor for CP, in silico, are presented.

Augmenting and assisting model elicitation tasks with 3D virtual world context metadata

Accurate process model elicitation continues to be a time consuming task, requiring skill on the part of the interviewer to extract explicit and tacit process information from the interviewee. Many errors occur in this elicitation stage that would be avoided by better activity recall, more consistent specification methods and greater engagement in the elicitation process by interviewees. Theories of situated cognition indicate that interactive 3D representations of real work environments engage and prime the cognitive state of the viewer. In this paper, our major contribution is to augment a previous process elicitation methodology with virtual world context metadata, drawn from a 3D simulation of the workplace. We present a conceptual and formal approach for representing this contextual metadata, integrated into a process similarity measure that provides hints for the business analyst to use in later modelling steps. Finally, we conclude with examples from two use cases to illustrate the potential abilities of this approach.

Mutagenic and cytotoxic activity of an isocoumarin (Paepalantine) isolated from Paepalanthus vellozioides

A new isocoumarin with antimicrobial activity was isolated from Paepalanthus vellozioides (a native Brazilian plant) and called paepalantine. This study was carried out to assess the mutagenic activity of this new agent in assays with Salmonella typhimurium TA100, TA98, and TA102 and in Chinese hamster ovary (CHO) cell cultures, as well as cytotoxicity to McCoy cells. Paepalantine caused a significant dose-dependent increase in the frequency of revertants in the three strains used in the assay, both with and without S9 mix, in concentrations varying from 2 to 128 mu g/ plate. The mutagenicity was confirmed in assays with CHO cells treated in the G(1), S, and G(2) phases of the cell cycle. There was an increase in the chromosomal aberration frequency, mainly in the G(2) phase. Furthermore, the mitotic index of the treated cultures (40,80, and 160 mu g/ml) was significantly lower, indicating cytotoxicity. The midpoint cytotoxicity values to McCoy cells by the neutral red (NR) and microculture tetrazolium (MTT) techniques resulted in a NR50, and MTT50 of 30 and 38 mu g/ml, respectively. Alterations to the paepalantine structure are suggested to reduce its mutagenic and cytotoxic activity in investigations for its antineoplasic potential (C) 1997 Wiley-Liss, Inc.

"Crop elicitation": approccio innovativo per la valorizzazione delle proprietà funzionali in leguminose da granella

Crop elicitation: innovative approach for the valorization of grain legume functional properties. In Italy grain legume cultivation has encountered a drastic decrease due to several causes (productive, economic, social). In this regard, studies aimed at the setting up of agronomic techniques able to guarantee high and constant in planta yields of health-promoting compounds may concur at re-launching legume production. In this context, 22 accessions of grain legumes (17 Phaseolus vulgaris, 3 Phaseolus coccineus, 1 Vigna unguiculata and 1 Glycine max genotypes) were screened with the aim of identifying genotypes rich in health beneficial phytochemicals (α-amylase inhibitors, α -glucosidase inhibitors, polyphenols) and with low anti-nutritional compounds (lectins). A wide variability was observed among investigated accessions. Four genotypes (Verdone, Kidney Cina, Roviotto and DG) showed a α -amylase inhibitory activity significantly higher (approximately 30% more) than all other tested accessions. The α -amylase inhibitory activity was not correlated neither with the protein nor with the polyphenol contents. Conversely, the α -glucosidase inhibitory activity was positively correlated with grain color and polyphenol content: dark-colored seeds had a mean inhibitory activity of 83.64 ± 22.07%, whereas light-colored seeds had mean values of 21.11 ± 9.36%. As regards the anti-nutritional compounds, out of all common bean accessions, only DG showed no erythro-agglutination activity (lectins). Preliminary experiments, performed in controlled environment, permitted to highlight that different germination conditions markedly affect the synthesis and accumulation of functional compounds in legume seedlings. Those findings were confirmed with field trials performed in two different locations (Bologna and Pisa), on two bean genotypes (Verdone and Zolfino), during the 2004-2005 cropping season. Results showed that the application of abiotic stresses (no fertilization and /or no irrigation) lead to a significant increase of flavonoids in grains, but a decrease (up to 50%) in legume yields was also observed. Crop elicitation, even if valuable for boosting health-promoting compound synthesis in crops, must necessary cope with economically acceptable crop yields.

Cutin Monomers and Surface Wax Constituents Elicit H2O2 in Conditioned Cucumber Hypocotyl Segments and Enhance the Activity of Other H2O2 Elicitors1

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface and cut segments were conditioned to develop competence for H2O2 elicitation. Alkaline hydrolysates of cutin from cucumber, tomato, and apple elicited H2O2 in such conditioned segments. The most active constituent of cucumber cutin was identified as dodecan-1-ol, a novel cutin monomer capable of forming hydrophobic terminal chains. Additionally, the cutin hydrolysates enhanced the activity of a fungal H2O2 elicitor, similar to cucumber surface wax, which contained newly identified alkan-1,3-diols. The specificity of elicitor and enhancement activity was further elaborated using some pure model compounds. Certain saturated hydroxy fatty acids were potent H2O2 elicitors as well as enhancers. Some unsaturated epoxy and hydroxy fatty acids were also excellent H2O2 elicitors but inhibited the fungal elicitor activity. Short-chain alkanols exhibited good elicitor and enhancer activity, whereas longer-chain alkan-1-ols were barely active. The enhancement effect was also observed for H2O2 elicitation by ergosterol and chitosan. The physiological significance of these observations might be that once the cuticle is degraded by fungal cutinase, the cutin monomers may act as H2O2 elicitors. Corrosion of cutin may also bring surface wax constituents in contact with protoplasts and enhance elicitation.

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.

Role of phosphorylation in elicitation of the oxidative burst in cultured soybean cells.

The oxidative burst is likely the most rapid defense response mounted by a plant under pathogen attack, and the generated oxidant species may be essential to several subsequent defense responses. In our effort to characterize the signal-transduction pathways leading to rapid H2O2/O2- biosynthesis, we have examined the role of protein phosphorylation in this resistance mechanism. K-252a and staurosporine, two protein-kinase inhibitors, were found to block the oxidative burst in a concentration-dependent manner. When added during H2O2 generation, the burst was observed to rapidly terminate, suggesting that continuous phosphorylation was essential for its maintenance. Importantly, phosphatase inhibitors (calyculin A and okadaic acid) were found to induce the oxidative burst in the absence of any additional stimulus. This may suggest that certain kinases required for the burst are constitutively active and that stabilization of the phosphorylated forms of their substrates is all that is required for burst activity. In autoradiographs of elicited and unstimulated cells equilibrated with 32PO4(3-), several phosphorylated polypeptide bands were revealed that could represent proteins essential for the burst.