Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.

Multiple stages of detergent-erythrocyte membrane interaction-A spin label study

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate- and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9 mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5 mM detergent. Between 3.2 and 10 mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10 mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs: the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5 mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111). (C) 2010 Elsevier B.V. All rights reserved.

Two New Ternary Complexes of Copper(II) with Tetracycline or Doxycycline and 1,10-Phenanthroline and Their Potential as Antitumoral: Cytotoxicity and DNA Cleavage

This paper reports on the synthesis and characterization of two new ternary copper(II) complexes: [Cu(doxy-cycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (1) and [Cu(tetracycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (2). These compounds exhibit a distorted tetragonal geometry around copper, which is coordinated to two bidentate ligands, 1,10-phenanthroline and tetracycline or doxycyline, a water molecule, and a perchlorate ion weakly bonded in the axial positions. In both compounds, copper(II) binds to tetracyclines`. via the oxygen of the hydroxyl group and oxygen of the amide group at ring A and to 1,10-phenanthroline via its two heterocyclic nitrogens. We have evaluated the binding of the new complexes to DNA, their capacity to cleave it, their cytotoxic activity, and uptake in tumoral cells. The complexes bind to DNA preferentially by the major groove, and then cleave its strands by an oxidative mechanism involving the generation of ROS. The cleavage of DNA was inhibited by radical inhibitors and/or trappers such as superoxide dismutase, DMSO, and the copper(I) chelator bathocuproine. The enzyme T4 DNA ligase was not able to relegate the products of DNA cleavage, which indicates that the cleavage does not occur via a hydrolytic mechanism. Both complexes present an expressive plasmid DNA cleavage activity generating single- and double-strand breaks, under mild reaction conditions, and even in the absence of any additional oxidant or reducing agent. In the same experimental conditions, [Cu(phen)(2)](2+) is approximately 100-fold less active than our complexes. These complexes are among the most potent DNA cleavage agents reported so far. Both complexes inhibit the growth of K562 cells With the IC(50) values of 1.93 and 2.59 mu mol L(-1) for compounds I and 2, respectively. The complexes are more active than the free ligands, and their cytotoxic activity correlates with intracellular copper concentration and the number of Cu-DNA adducts formed inside cells.

Proteome analysis of human dorsolateral prefrontal cortex using shotgun mass spectrometry

The prefrontal cortex executes important functions such as differentiation of conflicting thoughts, correct social behavior and personality expression, and is directly implicated in different neurodegenerative diseases. We performed a shotgun proteome analysis that included IEF fractionation, RP-LC, and MALDI-TOF/TOF mass spectrometric analysis of tryptic digests from a pool of seven human dorsolateral prefrontal cortex protein extracts. In this report, we present a catalog of 387 proteins expressed in these samples, identified by two or more peptides and high confidence search scores. These proteins are involved in different biological processes such as cell growth and/or maintenance, metabolism/energy pathways, cell communication/signal trarisduction, protein metabolism, transport, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism, and immune response. This analysis contributes to the knowledge of the human brain proteome by adding sample diversity and protein expression data from an alternative technical approach. It will also aid comparative studies of different brain areas and medical conditions, with future applications in basic and clinical research.

Proteome of the phytopathogen Xanthomonas citri subsp citri: a global expression profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enzymatic Hydrolysis of Sweet Lupin, Chickpea, and Lentil 11S Globulins Decreases their Antigenic Activity

We investigated the effects of treatments with the enzymes pepsin and trypsin on the in vitro immunological reactivity of the major globulins found in the seeds of sweet lupin, chickpea, and lentil. Polyclonal major globulin-specific antiserum was obtained by immunization of rabbits with a solution of the 11 S globulin of each legume. The globulins were hydrolyzed with pepsin and trypsin for 1, 5, 15, and 30 min. The native globulins and their hydrolysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify the polypeptide bands with antigenic activity, and the hypoantigenicity of the hydrolysates was analyzed by enzyme-linked immunosorbent assay. Our results show that enzymatic treatment of the major storage protein (11 S globulin) of sweet lupin, chickpea, and lentil with pepsin or trypsin lead to the formation of large amounts of short peptides and free amino acids that do not allow antibody binding, resulting in a weakened immunoreactivity.

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Myocardial dysfunction induced by food restriction is related to calcium cycling and beta-adrenergic system changes

Food restriction (FR) has been shown to promote myocardial dysfunction in rats. The aim of this study was to verify the participation of calcium and beta-adrenergic system on myocardial mechanical alteration in rats submitted to FR. Myocardial performance was studied in isolated left ventricular papillar muscle from young Wistar-Kyoto rats (WKY) submitted to FR or to control diet. The groups subjected to FR were fed 50% less food than the control group for 90 days. Mechanical function was studied in isometric contraction at post-rest contraction of 30 seconds (PRC), calcium chloride concentration 5.20 mM, and beta-adrenergic stimulation with isoproterenol 10(-6) M. FR decreased the body weight, and left and right ventricular weight. In basal condition (1.25 MM of calcium) time to peak tension (TPT) and time from peak tension to 50% relaxation (RT50) were greater in the FR group. Muscle function was. The same in both PRC groups. TPT decrease in both high calcium groups, more in FR rats; RT50 dropped only in FR animals. TPT decreased in both Isoproterenol groups, more intensely in the FR group. This result suggests that food restriction impairs myocardial performance and these changes may be attributed to alterations in the intracellular calcium cycling and beta-adrenergic system. (C) 2003 Elsevier B.V. All rights reserved.

Storage sites in seeds of Caesalpinia echinata and C. ferrea (Leguminosae) with considerations on nutrients flow

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The tricyclic antidepressants amitriptyline, nortriptyline and imipramine are weak antagonists of human and rat alpha(1B)-adrenoceptors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Orpotrin: A novel vasoconstrictor peptide from the venom of the Brazilian Stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97-105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms. (c) 2006 Elsevier B.V. All rights reserved.

Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Two new bradykinin-related peptides from the venom of the social wasp Protopolybia exigua (Saussure)

Two bradykinin-related peptides (Protopolybiakinin-I and Protopolybiakinin-II) were isolated from the venom of the social wasp Protopolybia exigua by RP-HPLC, and sequenced by Edman degradation method. Peptide sequences of Protopolybiakinin-I and Protopolybiakinin-II were DKNKKPIRVGGRRPPGFTR-OH and DKNKKPIWMAGFPGFTPIR-OH, respectively. Synthetic peptides with identical sequences to the bradykinin-related peptides and their biological functions were characterized. Protopolybiakinin-I caused less potent constriction of the isolated rat ileum muscles than bradykinin (BK). In addition, it caused degranulation of mast cells which was seven times more potent than BK. This peptide causes algesic effects due to the direct activation of B-2-receptors. Protopolybiakinin-II is not an agonist of rat ileum muscle and had no algesic effects. However, Protopolybiakinin-II was found to be 10 times more potent as a mast cell degranulator than BK. The amino acid sequence of Protopolybiakinin-I is the longest among the known wasp kinins. (c) 2006 Elsevier B.V. All rights reserved.

The intramandibular gland of leaf-cutting ants (Atta sexdens rubropilosa Forel 1908)

The eusociality developed in Hymenoptera and Isoptera is driven by an efficient interaction between exocrine glands and jointed appendages, both in close interaction with the environment. In this context, the mandible of ants plays an important role, since, in addition to being the main jointed appendage, it possess glandular functions. As an example we might name the two glands associated with the mandible: the mandibular and the intramandibular glands. The intramandibular gland is found inside the mandible and consists of a hypertrophied secretory epithelium and secretory cells in the mandible's lumen. The secretion of the secretory epithelium is liberated through intracuticular ducts that open at the base of hairs at the mandible's surface. The secretion of the intramandibular gland (epithelium and secretory cells) reacted positively to tests for the detection of polysaccharides and proteins, thus suggesting that it consists of glycoproteins. The ultrastructure of the secretor epithelium presents variations related to the developmental stage of the individual, showing a large number of ribosomes and microvilli close to the cuticle in young individuals, while in the older specimens it was possible to note the formation of ail intracellular reservoir. These variations of secretory epithelium, as also the interaction between the cellular groups inside the mandible, are important information about this gland in leaf-cutting ants. (c) 2005 Elsevier Ltd. All rights reserved.