Carlos Henrique Tomich de Paula da Silva

Digestive and absorptive sites along the midgut of the cotton seed sucker bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae)

Abstract The midgut of Dysdercus peruvianus is divided into three sections (V 1 −V 3 ), which are linked through V 4 to the hindgut. The midgut contents are reducing with a pH around 5.8. D. peruvianus saliva lacks digestive enzymes and apparently is used only to suspend seed material which in ingested, distending V 1 . Most midgut carbohydrase activities are found in V 1 , where is also observed a α-mannosidase and an aminopeptidase derived from the meal. The high glycosidase and low amylase activities found in V 1 are in agreement with the major and minor cotton seed content of saccharides and starch, respectively. Water and glucose absorption occur without dietary salt ions in V 1 . About 5 h after the filling of V 1 , V 2 is also filled. At V 2 , a cysteine-proteinae (pH optimum 5.5, M r 53,000) predominates and amino acids are absorbed. This absorption is activated by potassium and inhibited by sodium ions. Both midgut α-glucosidase and cysteine proteinase activities increase on feeding. As soon as V 1 empties, V 3 is filled. After a long stay (70–90 h) in V 2 and V 3 , the food passes quickly through V 4 into the hindgut. The results on D. peruvianus (Hemiptera: Pentatomomorpha) and literature data on Rhodnius prolixus (Hemiptera: Cimicomorpha) are used to infer the digestive physiology characteristics of the Heteroptera ancestor, and to discuss the major adaptations which occurred when the bugs moved from a sap sucking habit into a habit which included digestion of polymers.

Digestion in larvae of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis on α-amylases and oligosaccharidases

Abstract Determinations of carbohydrases, aminopeptidases and acid phosphatase in the larval midgut cells and in the luminal contents of Callosobruchus maculatus and Zabrotes subfasciatus have been carried out. The results showed that larvae of both species displayed similar distribution of digestive enzymes in the intestinal compartments. Most larval digestive enzyme activities were found in the luminal contents. Of the activities found in the midgut tissue, only aminopeptidase is predominant in the membrane fraction. Comparisons of activities recovered from a seed flour mass equivalent to the midgut mass showed that a high percentage of the luminal aminopeptidase activity and, to a lesser extent, α-galactosidase activity, can be derived from the seeds, whereas the other enzymes are produced by the insects. Activities against starch, maltose and maltodextrins were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on differences in elution profiles on hydrophobic chromatography and banding patterns in mildly denaturing electrophoresis, both species showed a multiplicity of glycosidases. The data suggest that the majority of carbohydrate digestion occurs in the midgut lumen, whereas protein digestion should take place partly in the lumen and partly at the cell surface. Larvae of Z. subfasciatus can modulate the levels of α-amylases and α-glucosidase in response to different diets. The complex of carbohydrases found is qualitatively appropriate to digest the free oligosaccharides and oligomaltodextrins produced by α-amylases from the starch granules of host seeds.

Organization, origin and function of the outer microvillar (perimicrovillar) membranes of Dysdercus peruvianus (Hemiptera) midgut cells

The midgut of Dysdercus peruvianus is divided into three main sections (V1–V3), and is linked through V4 to the hindgut. V1 cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes, which may be dilated in some places forming spheres, and have dead ends. The cytoplasmic side of the microvillar membrane is studded with small particles (portasomes), putatively involved in creating ionic gradients used in the cotransport of organic compounds. The basal membrane invaginates forming narrow and ramified channels with associated mitochondria. V2, V3 and V4 cells are similar to V1 cells, except for less developed basal infoldings. V4 cells lack particles associated to the cytoplasmic side of the microvillar membranes and morphological features associated with protein secretion. V3 and V4 cells show bacteria associated to the microvilli. These morphological features agree with physiological data showing that V1 absorbs water, V2 and V3 absorb amino acids and no digestion occurs in V4. α-Glucosidase, a perimicrovillar membrane marker, was immunolocalized in V1 cells. The data suggest that perimicrovillar membranes are formed in Golgi areas, migrate as the internal membrane of double membrane vesicles, which finally fuse at the cell apex—the outer vesicle membrane with the microvillar membrane and the inner vesicle membrane with the perimicrovillar membrane.

Current topics in computer‐aided drug design

The addition of computer-aided drug design (CADD) technologies to the research and drug discovery approaches could lead to a reduction of up to 50% in the cost of drug design. Designing a drug is the process of finding or creating a molecule which has a specific activity on a biological organism. Development and drug discovery is a time-consuming, expensive, and interdisciplinary process whereas scientific advancements during the past two decades have altered the way pharmaceutical research produces new bioactive molecules. Advances in computational techniques and hardware solutions have enabled in silico methods to speed up lead optimization and identification. We will review current topics in computer-aided molecular design underscoring some of the most recent approaches and interdisciplinary processes. We will discuss some of the most efficient pathways and design.

Genomic organisation and transcription characterisation of the gene encoding Leishmania (Leishmania) amazonensis arginase and its protein structure prediction.

The genomic organisation of the gene encoding Leishmania (Leishmania) amazonensis arginase as well as its flanking regions were characterised. The size of the transcribed RNA was determined, allowing us to map the genomic sites signalling for RNA trans-splicing and putative polyadenylation regions. The general organisation was compared with genes encoding other proteins already described in organisms of the Trypanosomatid family. The complete nucleotide sequence of the arginase open reading frame was obtained and the three-dimensional structure of the enzyme was inferred by a computational analysis of the deduced amino acid sequence, based on the established crystal structure described for Rattus norvergicus arginase. The human liver arginase sequence was analysed in the same way and the comparison of the presumed structure of both the Leishmania and human enzymes identified some differences that may be exploited in chemotherapeutic studies.

Two-photon absorption in azoaromatic compounds

Abstract We report on the two-photon absorption (2PA) cross-section for eight azoaromatic compounds in dimethyl sulfoxide solutions, using the Z-scan technique with femtosecond laser pulses at 775 nm. The 2PA cross-sections of these molecules are of the order of (1–6)×10 −50 cm 4 s photon −1 . A correlation was established between the molecular structure and the two-photon absorption process, which makes it possible to develop molecular design strategies to create structures with suitable two-photon absorption coefficients.

An α-glucosidase from perimicrovillar membranes of Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) midgut cells. Purification and properties

Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) has a major α-glucosidase bound to perimicrovillar membranes, which are the lipoprotein membranes ensheathing the midgut cell microvillar membranes in hemipterans. The enzyme was solubilized in detergent and purified to homogeneity by means of affinity chromatography on Concanavalin A-Sepharose, ion-exchange on Mono Q and preparative polyacrylamide gel electrophoresis (PAGE). The yield, purification factor and final specific activity were, respectively: 3.5%, 50-fold, 7.6 U/mg of protein. The α-glucosidase is a glycoprotein with a pH optimum of 5.0. Mr values were 61,000 (SDS-PAGE), 120,000 (gel filtration), 130,000 (ultracentrifugation), or 431,000 (electrophoresis in native conditions). The data suggest that the α-glucosidase occurs in vivo as dimers and, during electrophoresis, as octamers. Taking into account kcat/Km ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltotetraose. Using p-substituted phenyl α-glucosides as substrates, it was shown that aglycone leaving is rate limiting and that the reaction constant p is negative, suggesting the intermediary formation of a carbonium ion in the reaction path. Erythritol, δ-gluconolactone and Tris are simple linear competitive inhibitors of the α-glucosidase. Experiments involving competition between substrates suggested that the enzyme operates in accordance with rapid-equilibrium kinetics, hydrolyzing maltose, p-nitrophenyl-α-d-glucoside, sucrose and turanose at the same active site. Evidence suggests that the active site may have five sub-sites.

Computer-aided Drug Design of Novel PLA2 Inhibitor Candidates for Treatment of Snakebite

Abstract Phospholipases A2 (PLA2) are enzymes commonly found in snake venoms from Viperidae and Elaphidae families, which are major components thereof. Many plants are used in traditional medicine as active agents against various effects induced by snakebite. This article presents the PLA2 BthTX-I structure prediction based on homology modeling. In addition, we have performed virtual screening in a large database yielding a set of potential bioactive inhibitors. A flexible docking program was used to investigate the interactions between the receptor and the new ligands. We have performed molecular interaction fields (MIFs) calculations with the phospholipase model. Results confirm the important role of Lys49 for binding ligands and suggest three additional residues as well. We have proposed a theoretically nontoxic, drug-like, and potential novel BthTX-I inhibitor. These calculations have been used to guide the design of novel phospholipase inhibitors as potential lead compounds that may be optimized for future treatment of snakebite victims as well as other human diseases in which PLA2 enzymes are involved.

Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera : Bruchidae) and the induction of α-amylases in response to different diets

Zabrotes subfasciatus larvae possess three alpha-amylase isoforms as determined by in gel assays following SDS-PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of alpha-amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor alpha-amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different alpha-amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.

Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (hemiptera: pyrrhocoridae) midgut cells

Abstract The midgut of Dysdercus peruvianus is divided into four sections (V1-V4). All the cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes with dead ends. Subcellular fractionation of V1 and V2 tissue in isotonic and hypotonic conditions showed that α-glucosidase is associated with membranous structures larger than those associated with β-glucosidase. The α/β-glucosidase activity ratio is 34 ± 4 in V1 tissue and 170 ± 10 in membranes recovered from the V1 luminal contents. These membranes are resolved in sucrose gradients into low density (1.087 ± 0.001 g/cm3) α-glucosidase-carrying membranes (α/β-glucosidase activity ratio of 330±30) and high density (1.132 ± 0.002g/cm3) β-glucosidase-carrying-membranes. Low-density membranes have 1090 ± 60 μg lipid/mg protein and apparently are not contaminated by high-density ones (electron micrographs). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that membranes recovered from V1 luminal contents are composed mainly of a-glucosidase-rich membranes. The data suggest that α-glucosidase-rich membranes are perimicrovillar membranes which may be partly lost into luminal contents on dissection, with densities and lipid/protein ratios similar to that of myelin sheaths, in accordance with previous freeze-fracture data. β-Glucosidase-rich membranes are probably microvillar membranes with densities increased by the presence of associated portasomes.