Análogos sintéticos da cheferina I: interação com íons metálicos divalentes e o seu efeito na internalização celular e nas atividades anticandida e candidacida / Synthetic analogues of Shepherin I: interaction with metal divalent ions and their effect on cellular internalization and on anticandidal/candidacidal activitie

O desenvolvimento de resistência antimicrobiana e a consequente seleção de microrganismos multirresistentes consolidam-se como grandes ameaças à saúde global. Neste contexto, a busca por novas drogas antimicrobianas/microbicidas é fundamental e compostos como os peptídeos antimicrobianos (AMPs) tornaram-se alvos atraentes. Os AMPs são compostos químicos de massa molar média e grande diversidade estrutural, produzidos por todos os seres vivos e com capacidade de inibir o crescimento de e/ou matar microrganismos. O AMP Cheferina I (Chef I) foi isolado das raízes de Capsella bursa-pastoris e é resultado da proteólise de uma proteína da família das proteínas ricas em glicina, que em plantas estão relacionadas às funções de defesa e cicatrização. O nosso grupo de pesquisa foi pioneiro no desenvolvimento e estudo de análogos truncados amidados deste AMP atípico rico em glicina (67,9%) e histidina (28,6%), que se mostraram ativos frente às diferentes cepas de Candida e a S. cerevisiae pela internalização/ação celular acompanhada de manutenção da integridade da membrana plasmática; o análogo amidado (Chef Ia) e o análogo marcado com 5(6)-carboxifluoresceína/FAM (FAM-Chef Ia) tiveram as suas atividades antifúngicas potencializadas por íons Zn2+. Este trabalho deu continuidade ao estudo do efeito dos íons metálicos divalentes Zn2+, Cu2+, Ca2+ e Mg2+ nas atividades anticandida/fungistática e candidacida/fungicida a diferentes pHs e forças iônicas, estruturas e localizações intracelulares destes análogos. Os resultados na ausência de íons em pH 5,1 revelaram maior atividade do análogo fluorescente em relação à do não fluorescente. Neste mesmo pH, as atividades anticandida e candidacida de Chef Ia foram influenciadas negativamente pelos íons Ca2+ e Mg2+ (2-4 vezes) enquanto que, na presença de íons Zn2+ as atividades anticandida de ambos os análogos foram aumentadas (Chef Ia: 8-64 vezes; FAM-Chef Ia: 4-32 vezes). Os íons Cu2+ aumentaram a atividade anticandida de Chef Ia (2-4 vezes), mas não a do análogo fluorescente, mas as atividades candidacidas de ambos foram melhoradas (Chef Ia: 2-8 vezes; FAM-Chef Ia: 2 vezes). Em pH 5,1, os íons Zn2+ mantiveram a atividade anticandida de Chef Ia em alta força iônica, mas só FAM-Chef Ia exibiu atividade candidacida. Em pH 7,4 ambos análogos foram inativos em baixa e alta forças iônicas na ausência e presença de Zn2+ ou Cu2+. As maiores porcentagens de folhas-ß-antiparalelas e dobras foram observadas no espectro de DC de Chef Ia em pH 7,4, sendo que aqueles registrados em pH 5,1 e 7,4 em presença de íons Zn2 e Cu2+ indicaram a formação de quelatos estruturalmente distintos. Ambos os peptídeos são bioquelantes em potencial, sendo as proporções peptídeo: íon obtidas as seguintes: FAM-Chef Ia = 1:2 para Cu2+, 1:10 para Zn2+; Chef Ia = 1:1 para Cu2+. A análise da internalização celular de FAM-Chef Ia permitiu a suposição de dois mecanismos de internalização (translocação direta e endocitose), sendo que nas células vivas a presença de Zn2+ afetou negativamente a translocação direta (p 0,0343) e potencializou a endocitose (p 0,0002)

The development of antimicrobial resistance and the consequent selection of multiresistant microorganisms have become major threats to global health. In this context, the search for new antimicrobial/microbicidal drugs is crucial and the antimicrobial peptides (AMPs) have been seen as attractive targets. AMPs are chemical compounds of medium molecular mass and high structural diversity produced by all living beings, capable of inhibiting the growth of microorganisms and killing them. The AMP Shepherin I (Shep I) was isolated from the roots of Capsella bursa-pastoris, being a bioactive peptide encrypted in a glycine-rich protein from a family that in plants are strictly related to defense and healing functions. Our research group has pioneered the development and study of amidated truncated analogues of this atypical glycine- (67.9%) and histidine-rich (28.6%) AMP, which has shown activity against different strains of Candida and S. cerevisiae through cellular internalization with maintenance of the plasma membrane integrity. The amide analogue (Chef Ia) and its fluorescent analog labeled with 5 (6) - carboxyfluorescein / FAM (FAM-Chef Ia) had their antifungal activities potentiated by Zn2+ ions, so the present work continued examining the effect of the divalent metallic ions Zn2+, Cu2+, Ca2+ and Mg2+ on the anticandidal/fungistatic and candidacidal/fungicide activities at different pHs and ionic forces, structures and intracellular locations of these analogues. The results in the absence of those ions at pH 5.1 revealed that the fluorescently labelled analog was more potent than the nonfluorescent. At the same pH, Shep Ia anticandidal and candidacidal activities were negatively influenced by Ca2+ and Mg2+ ions (2-4 fold), whereas in the presence of Zn2+ ions the anticandidal activities of both analogues were increased (Shep Ia: 8-64 fold, FAM- Shep Ia: 4-32 fold). Cu2+ ions increased Shep Ia anticandidal activity (2-4 fold) but not that of FAM-Shep Ia, nevertheless, the candidacidal activities of both analogues were increased (Shep Ia: 2-8 fold, FAM-Shep Ia: 2 fold). Also at pH 5.1, the Zn2+ ions helped retaining the anticandidal activity of Shep Ia at high ionic strength, although only FAM-Shep Ia exhibited candidacidal activity. At pH 7.4 both analogues were inactive at low and high ionic strengths in the absence or presence of Zn2+ or Cu2+. The highest percentages of antiparallel ß-sheet and turns were observed in Shep Ia CD spectrum at pH 7.4, while those recorded at pH 5.1 and 7.4 in the presence of Zn2+ or Cu2+ ions indicated the formation of structurally different chelates. Both peptides are potential biochelates, with the following peptide:ion ratios: FAM-Shep Ia = 1: 2 for Cu2+, 1:10 for Zn2+; Shep Ia = 1: 1 for Cu2+. The analysis of the cellular internalization of FAM-Chef Ia allowed the assumption of two mechanisms of internalization (direct translocation and endocytosis) and in the living cells the presence of Zn2+ negatively affected the direct translocation (p 0.0343) and potentiated endocytosis (p 0.0002)

Phytochemical Constituents of Capsella bursa-pastoris and Their Anti-inflammatory Activity

Phytochemical investigation of 80% MeOH extract of the aerial parts of Capsella bursa-pastoris yielded fourteen compounds (1 – 14). The structures of the compounds were elucidated by spectroscopic methods to be methyl-1-thio-β-D-glucopyranosyl disulfide (1), 10-methylsulphinyl-decanenitrile (2), 11-methyl-sulphinyl-undecanenitrile (3), 1-O-(lauroyl)glycerol (4), phytene-1, 2-diol (5), (3S,5R,6S,7E)-5,6-epoxy-3-hydroxy-7-megastigmen-9-one (6), loliolide (7), β-sitosterol (8), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (9), 1-feruloyl-β-D-glucopyranoside (10), pinoresinol-4′-O-β-D-glucopyranoside (11), luteolin (12), quercetin-3-O-β-D-glucopyranoside (13), and luteolin 6-C-β-glucopyranoside (14). Although compound 1 was reported as synthetic compound, 1 was first isolated from natural source. NMR spectral data assignments of 1, 2 and 3 were reported for the first time, and compounds 1 – 14 were for the first time reported from this plant source. The anti-inflammatory effects of 1 – 14 were evaluated in lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cells. Compounds 12 exhibited strong inhibitory effects on nitric oxide production in LPS-activated BV-2 cells with IC50 values of 9.70 µM.

Molecular characterization and expression pattern of a novel cadmium resistance gene of tobacco / Caracterização e padrão de expressão molecular de um novo gene de fumo para resistência ao cádmio

Cadmium (Cd) of tobacco is a pollutant that is extremely toxic to the health of humans. Protein plant cadmium resistance 8 gene has been characterized to increase the plant Cd resistance. In present experiment, the complete mRNA sequence of tobacco protein plant cadmium resistance 8 gene was amplified using the rapid amplification of cDNA ends methods. The full-length tobacco protein plant cadmium resistance 8 gene mRNA was 887bp containing an 555 bp open reading frame, which encodes a protein of 184 amino acids. BLAST analysis revealed that tobacco protein plant cadmium resistance 8 protein shares high homology with the protein plant cadmium resistance 8 of potato (81%), Lycopersicon esculentum (80%), Eutrema salsugineum (60%), Capsella rubella (58%) and thale cress (57%). Results also showed that tobacco protein plant cadmium resistance 8 gene has a closer genetic relationship with the protein plant cadmium resistance 8 gene of Lycopersicon esculentum. The expression profile was studied and the results indicated that tobacco protein plant cadmium resistance 8 gene was highly expressed in root, moderately expressed in stem, and hardly expressed in flower and leaf. These results established the primary foundation of utilizing tobacco protein plant cadmium resistance 8 gene to decrease the cadmium content of tobacco and benefit the health of humans in the future.

Cádmio (Cd) oriundo do fumo é um poluente que é extremamente tóxico a saúde humana. Um gene encontrado na planta de fumo que codifica uma proteína para resistência (gene 8) tem sido caracterizado para aumentar a resistência do fumo na absorção do elemento Cádmio (Cd).No experimento realizado a seqüência do mRNA de fumo que codifica uma proteína foi amplificada usando métodos de amplificação de CDNAs. A proteína completa codificada pelo gene 8 apresenta um mRNA com 887 pb com uma fita de leitura de 555 pb, a qual codifica 184 aminoácidos. A análise de BLAST demonstrou uma homologia de 81 % com o gene 8 da batateira, de 80 % com o tomateiro (Lycopersicon esculentum), 60 % para Eutrema salsugineum , 58 % para Capsella rubella e 57 % para o agrião. A proteína expressada pelo gene 8 para resistência do fumo a absorção do Cádmio também apresenta um forte relacionamento genético com a proteína expressa pelo gene 8 do tomateiro (Lycopersicon esculentum). Os perfis da expressão protéica para a proteína oriunda do gene 8 do fumo foi de grande magnitude em raiz, moderadamente expressa no caule e de difícil expressão nas flores e nas folhas. Estes resultados obtidos fundamentam o uso deste gene 8 de fumo para resistência ao Cádmio com o propósito de reduzir o teor de cádmio na planta de fumo e com reflexos benéficos para a saúde humana no futuro.

Anti-Inflammatory and Anti-Superbacterial Properties of Sulforaphane from Shepherd's Purse

Shepherd's purse, Capsella bursa-pastoris (L.) Medik., has been considered a health food for centuries in Asia and is known to contain the isothiocyanate compound sulforaphane. In this study, we evaluated the anti-inflammatory and antibacterial properties of a sulforaphane-containing solution (SCS) isolated from shepherd's purse. SCS had significant anti-inflammatory activity indicated by the decreased levels of nitric oxide (NO), cytokines (interleukin 1beta [IL-1beta], IL-6, and IL-10), and prostaglandin E2 (PGE2) in lipopolysaccharide-stimulated RAW 264.7 murine macrophages. In addition, SCS decreased the inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) levels, which confirmed the anti-inflammatory activity of SCS. Further, SCS inhibited vancomycin-resistant enterococci (VRE) and Bacillus anthracis. The minimal inhibitory concentration was 250 microg/ml for VRE and 1,000 microg/ml for B. anthracis. Taken together, these data indicate that SCS has potential anti-inflammatory and anti-superbacterial properties, and thus it can be used as a functional food or pharmaceutical.

Isolation and molecular characterization of a cax gene from Capsella bursa-pastoris

A new cation exchangers (CAXs) gene was cloned and characterized from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence of cax from C. bursa-pastoris (designated as Cbcax51) was 1754 bp containing a 1398 bp open reading frame encoding a polypeptide of 466 amino-acid residues with a calculated molecular mass of 50.5 kDa and an isoelectric point of 5.69. The predicted CbCAX51 contained an IMP dehydrogenase/GMP reductase domain, two Na+/Ca2+ exchanger protein domains. Comparative and bioinformatics analyses revealed that CbCAX51 showed extensive homology with CAX from other plant species. The expression analysis by different treatments indicated that Cbcax51 could be activated by cold triggering and was related to the cold acclimation process, but its expression is regulated negatively by drought and not affected by ABA or salt.

Produtividade e valor nutritivo da Brachiaria brizantha cv. Marandu em um sistema silvipastoril / Productivity and nutritional value of Brachiaria brizantha cv. Marandu in a silvopastoral system

O experimento foi realizado em sistema silvipastoril (Ssp) localizado no bioma de Cerrado, no município de Lagoa Santa, Minas Gerais, no ano agrícola de 2004-2005. Avaliou-se a influência do Ssp composto pela arbórea bolsa-de-pastor (Zeyheria tuberculosa) sobre os parâmetros produção, atributos morfogênicos, composição bromatológica e valor nutritivo da gramínea Brachiaria brizantha cv. Marandu, e os efeitos sobre as condições microclimáticas. O delineamento experimental foi em blocos ao acaso, com cinco blocos (épocas de corte), dois tratamentos (T1 - B. brizantha cv. Marandu sombreada com a Z. tuberculosa e T2 - B. brizantha cv. Marandu, a pleno sol) e três repetições (parcelas). O sombreamento proporcionado pela Z. tuberculosa, apesar de reduzir a produção de matéria seca (MS) da gramínea, favoreceu o aumento do teor de proteina bruta (PB) e não alterou a produção de PB/ha e nem afetou a concentração de FDN. Os teores de FDA foram mais altos que os encontrados a pleno sol, o que provavelmente resultou em menor degradabilidade in vitro da MS e da matéria orgânica, não alterando a degradabilidade efetiva

The experiment was carried out in a Silvopastoral system (Sps) located in Brazilian Savannah, Lagoa Santa county, Minas Gerais State, during the agricultural year of 2004-2005. It was studied the influence of Sps formed by "Bolsa de Pastor" (Zeyheria tuberculosa) on the production, morphogenic attributes, bromatological composition and nutritional value of the graminaceous Brachiaria brizantha cv. Marandu as well as on the microclimatic aspects. The Sps was established in 1982, in a red-yellow latossol by the natural regeneration process. The experiment was based on a randomized block design, with 5 blocks (cutting times), 2 treatments (T1 - B. brizantha cv. Marandu shaded by Z. tuberculosa and T2 - B. brizantha cv. Marandu at open sky and three repetitions (plots). Although the DM production of the graminaceous in the Sps with Z. tuberculosa has reduced the level of crude protein (CP) has increased and the production of CP per ha and the Sps NDF concentration have remained unchanged. However, the levels of ADF were higher in the shaded area, which probably resulted in lower in vitro DM and organic matter degradability, but similar effective DM degradability

In vitro Effects of Plant Extracts, and Phytohormones on Mycelial Growth of Anthracnose Fungi

Water extracts of six plants, such as Allium sativum, A. cepa, Zingiber officinale, Platycodon grandiflorum, Oenanthe javanica, and Capsella brusapastoris, were tested in vitro for inhibitory activity against mycelial growth of anthracnose fungi, Colletotrichum gloeosporioides, C. dematium, and C. coccodes. Among the plant extracts, an Allium sativum extract has good inhibitory effects in all the fungi. Four phytohormones namely, IAA (indole-3-acetic acid), NAA (a-Naphthyl acetic acid), 2,4-D (2,4-Dichloro phenoxy acetic acid) and BAP (Benzyl adenine purine) were used to find out the role over mycelial growth of these fungi. All the concentrations of BAP have good inhibitory effect against mycelial growth of these fungi than that of other tested plant hormones.