Emilio de Castro Miguel

Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties.

BACKGROUND A growing number of cysteine-rich antimicrobial peptides (AMPs) have been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides, which include lipid transfer proteins (LTPs), play an important role in the protection of plants against microbial infection. METHODS Peptides from Coffea canephora seeds were extracted in Tris-HCl buffer (pH 8.0), and chromatographic purification of LTP was performed by DEAE and reverse-phase HPLC. The purified peptide was submitted to amino acid sequence, antimicrobial activity and mammalian α-amylase inhibitory analyses. RESULTS The purified peptide of 9kDa had homology to LTPs isolated from different plants. Bidimensional electrophoresis of the 9kDa band showed the presence of two isoforms with pIs of 8.0 and 8.5. Cc-LTP(1) exhibited strong antifungal activity, against Candida albicans, and also promoted morphological changes including the formation of pseudohyphae on Candida tropicalis, as revealed by electron micrograph. Our results show that Cc-LTP(1) interfered in a dose-dependent manner with glucose-stimulated, H(+)-ATPase-dependent acidification of yeast medium and that the peptide permeabilized yeast plasma membranes to the dye SYTOX green, as verified by fluorescence microscopy. Interestingly, we also showed for the first time that the well characterized LTP(1) family, represented here by Cc-LTP(1), was also able to inhibit mammalian α-amylase activity in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE In this work we purified, characterized and evaluated the in vitro effect on yeast of a new peptide from coffee, named Cc-LPT1, which we also showed, for the first time, the ability to inhibit mammalian α-amylase activity.

Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)

(Ultraestrutura da fase secretora e da senescente dos coleteres de Bathysa gymnocarpa e B. stipulata (Rubiaceae)). Coleteres sao estruturas secretoras formadas por um eixo parenquimatico que inclui feixes vasculares, circundado por uma camada de celulas epidermicas secretoras em palicada. Em estudos sobre a estrutura dos coleteres tem sido observada a ultraestrutura das celulas secretoras, mas nao discriminam as fases secretora e senescente. Geralmente, em celulas secretores de mucilagem como os coleteres, o reticulo endoplasmatico e o complexo de Golgi estao envolvidos na producao e no transporte da secrecao. Neste estudo, foram determinadas duas fases baseadas na estrutura dos coleteres de Bathysa gymnocarpa K. Schum. and B. stipulata (Vell.) C. Presl. (Rubiaceae): a fase secretora e a fase senescente. Amostras foram coletadas e processadas utilizando tecnicas usuais de microscopia optica e eletronica. Os coleteres estudados sao constituidos por uma camada epidermica em palicada e um eixo central parenquimatico com tracos vasculares raros. Durante a fase secretora, as celulas epidermicas se apresentam com o citoplasma denso, pequenos vacuolos, reticulo endoplasmatico liso e rugoso evidente e complexo de Golgi proximo a grandes vesiculas. Durante a fase senescente, as celulas epidermicas apresentaram o sistema endomembranar desorganizado. Nenhuma organela intacta ou vesicula foi observada. A parede celular mais externa exibiu camadas similares as observadas durante a fase secretora. A fase senescente e facilmente definida pela morfologia do coleter, mas nao e bem definida em nivel subcelular nas celulas secretoras. Nossa investigacao sugere que a morte celular programada se inicia na fase secretora. Contudo, mais evidencias sao necessarias para avaliar esse fenomeno.

Ultrasound processing to enhance drying of cashew apple bagasse puree: Influence on antioxidant properties and in vitro bioaccessibility of bioactive compounds

The present study has evaluated the effects of power ultrasound pre-treatment on air-drying and bioactive compounds of cashew apple bagasse. The sonication induced the disruption of cashew bagasse parenchyma, which resulted in lower resistance to water diffusion, less hysteresis, and increased rehydration rate. The processing did not affect the lignocellulose fibers or the sclerenchyma cells. For sonicated samples, water activity reached values below 0.4, after 2h of drying, which is appropriate to prevent bacterial and fungi growth. The sorption isotherms of cashew apple bagasse presented sigmoid-shape for all samples and followed the type II according to BET classification. Sonicated cashew apple bagasse showed higher antioxidant activity, higher total phenolic compounds (TPC) and higher vitamin C content when compared to the non-sonicated sample. The increase in TPC and vitamin C contributed to the product antioxidant activity. A slight reduction on Vitamin C bioaccessibility was observed, but the TPC bioaccessibility has increased. Sonication reduced the quality loss of conventional drying treatments improving the quality of the dried product.

Purification, partial characterization and immobilization of a mannose-specific lectin from seeds of Dioclea lasiophylla mart.

Lectin from the seeds of Dioclea lasiophylla (DlyL) was purified in a single step by affinity chromatography on a Sephadex® G-50 column. DlyL strongly agglutinated rabbit erythrocytes and was inhibited by monosaccharides (D-mannose and α-methyl-d-mannoside) and glycoproteins (ovalbumin and fetuin). Similar to other Diocleinae lectins, DlyL has three chains, α, β and γ, with mass of 25,569 ± 2, 12,998 ± 1 and 12,588 ± 1 Da, respectively, and has no disulfide bonds. The hemagglutinating activity of DlyL was optimal in pH 8.0, stable at a temperature of 70 °C and decreased in EDTA solution, indicating that lectin activity is dependent on divalent metals. DlyL exhibited low toxicity on Artemia sp. nauplii, but this effect was dependent on the concentration of lectin in solution. DlyL immobilized on cyanogen bromide-activated Sepharose® 4B bound 0.917 mg of ovalbumin per cycle, showing the ability to become a tool for glycoproteomics studies.

Accumulation of organotins in seafood leads to reproductive tract abnormalities in female rats

Organotins (OTs) are environmental contaminants used as biocides in antifouling paints that have been shown to be endocrine disrupters. However, studies evaluating the effects of OTs accumulated in seafood (LNI) on reproductive health are particularly sparse. This study demonstrates that LNI leads to impairment in the reproductive tract of female rats, as the estrous cycle development, as well as for ovary and uterus morphology. Rats were treated with LNI, and their reproductive morphophysiology was assessed. Morphophysiological abnormalities, such as irregular estrous cycles, abnormal ovarian follicular development and ovarian collagen deposition, were observed in LNI rats. An increase in luminal epithelia and ERα expression was observed in the LNI uteri. Together, these data provide in vivo evidence that LNI are toxic for reproductive morphophysiology, which may be associated with risks to reproductive function.

Leaf structure, microanalysis and characterization of the latex protein profile of Pachystroma longifolium (Nees) I.M. Jonhst. (Euphorbiaceae) in a seasonally dry Atlantic Forest

Pachystroma longifolium is an evergreen species of Euphorbiaceae that occurs in tabuleiro forest, a type of lowland Atlantic Forest. The site chosen for this study was a fragment of tabuleiro forest that has a history of being selectively logged for timber. P. longifolium is very successful at colonizing disturbed areas in this forest fragment in comparison with preserved fragments. In the present work, which was based on using different microscopy (electron and light microscopy, X-ray analysis) and biochemistry techniques, we describe the leaf and latex traits of P. longifolium and their role as defense mechanisms against desiccation and herbivory. Our results suggest the richness of P. longifolium populations, in the disturbed forest area studied, is primarily a consequence of unpalatable leaves because of an abundance of calcium oxalate crystals in the subjacent epidermis; the presence of phenolic compounds in the adaxial surface; and a laticifer system that contains a complex mixture of proteins, which provides resistance to herbivores. In addition, this species is resistant to desiccation during dry periods because of an extensive amount of wax that occurs on the outer cell walls of the epidermis, and its ability to retain water because of a biseriate epidermis.

Hydrochar as protein support: preservation of biomolecule properties with non-covalent immobilization

In this work, the ConBr lectin was non-covalently immobilized onto hydrochar (HC). This carbonaceous material was produced by the hydrothermal carbonization of glucose and then put to interact with the lectin, aiming to immobilize the biomolecule via electrostatic interactions. Samples obtained after the interaction were characterized by CHNS elemental analysis, scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR). FTIR results from the conjugated sample identified the presence of NH2+ and NH3+ groups of the protein and COO− groups of the HC, indicating the occurrence of electrostatic interaction between the biomolecule and the support. Furthermore, the immobilization experiment was also performed using ConBr lectin marked with fluorescein isothiocyanate to assess the immobilization on the hydrochar using fluorescence emission analysis. Hemagglutination tests revealed that even after the conjugation with the HC, the agglutinating property of lectin toward erythrocytes (red blood cells) was preserved. Finally, our results indicate that non-covalent interactions represent an efficient mechanism for protein immobilization on the HC while maintaining the protein structure and its biological activity.

MORFOANATOMIA DE Ricinus communis L. (Euphorbiaceae) EXPOSTAS A ESTRESSE SALINO

A mamoneira e uma planta da familia Euphorbiacea e encontra-se amplamente distribuida em todo o territorio brasileiro, onde se adaptou com grande facilidade. Sua capacidade de adaptacao a variadas condicoes e sua resistencia ao deficit hidrico, permitem seu desenvolvimento em solos pouco ferteis e em condicoes adversas para a maior parte das plantas. Sua cultura e considerada bastante rustica, no entanto, e sensivel as caracteristicas fisicas e quimicas do solo como granulometria, pH e presenca de sais. Neste contexto, o objetivo desse trabalho foi caracterizar as alteracoes morfoanatomicas apresentadas por plantas de Ricinus communis L., cultivar Mirante 10, expostas a estresse salino. Para tanto, as sementes foram postas para germinar em dois substratos, areia e terra, contendo diferentes concentracoes de sal. Os parâmetros avaliados foram o comprimento das plântulas, percentual de materia seca, area foliar e densidade estomatica. Os dados anatomicos foram obtidos atraves de cortes a mao livre de caule, folha e raiz, clarificados, observados e fotografados em microscopio optico com câmera digital acoplada. Os resultados mostraram que as plantas de Ricinus communis L. respondem de forma diferenciada aos niveis salinos e substratos, com respostas mais fortes encontradas nas mudancas anatomicas, principalmente nos tecidos radiculares. Os dois substratos testados influenciaram diretamente na emergencia e estabelecimento destas plantas a condicao de salinidade do solo.