Predição de sítios de ligação para a cisplatina e transplatina baseada em ligações de hidrogênio

Pós-graduação em Biofísica Molecular - IBILCE

Structure of importin-a from a filamentous fungus in complex with a classical nuclear localization signal

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

Chemotactic signal transduction and phosphate metabolism as adaptive strategies during citrus canker induction by Xanthomonas citri

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

Binding analysis of Bacillus thuringiensis Cry1 proteins in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae)

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

Modulation of synaptic transmission in hippocampal CA1 neurons by a novel neurotoxin (beta-pompilidotoxin) derived from wasp venom

We examined the effects of beta-pompilidotoxin (beta-PMTX), a neurotoxin derived from wasp venom. on synaptic transmission in the mammalian central nervous system (CNS). Using hippocampal slice preparations of rodents, we made both extracellular and intracellular recordings from the CA1 pyramidal neurons in response to stimulation of the Schaffer collateral/commissural fibers. Application of 5-10 muM beta-PMTX enhanced excitatory postsynaptic potentials (EPSPs) but suppressed the fast component of the inhibitory postsynaptic potentials (IPSPs). In the presence of 10 muM bicuculline, beta-PMTX potentiated EPSPs that were composed of both non-NMDA and NMDA receptor-mediated potentials. Potentiation of EPSPs was originated by repetitive firings of the prosynaptic axons, causing Summation of EPSPs. In the presence of 10 muM CNQX and 50 muM APV, beta-PMTX suppressed GABA(A) receptor-mediated fast IPSPs but retained GABA(B) receptor-mediated slow IPSPs. Our results suggest that beta-PMTX facilitates excitatory synaptic transmission by a presynaptic mechanism and that it causes overexcitation followed by block of the activity of some population of interneurons which regulate the activity of GABA(A) receptors. (C) 2001 Published by Elsevier B.V. Ireland Ltd and the Japan Neuroscience Society.

Role of α1-, and α2- and β-adrenoceptors of the median preoptic area on the water intake, renal excretion, and arterial pressure induced by ANG II

The present experiments were conducted to investigate the role of the α1-, α2- and β-adrenergic receptors of the median preoptic area (MnPO) on the water intake and urinary electrolyte excretion, elicited by central injections of angiotensin II (ANG II). Prazosin (an α1-adrenergic receptor antagonist) and yohimbine (an α2-adrenergic receptor antagonist) antagonized the water ingestion, Na +, K +, and urine excretion induced by ANG II. Administration of propranolol, a β-adrenergic receptor antagonist increased the Na +, K +, and urine excretion induced by ANG II. Previous treatment with prazosin and yohimbine reduced the pressor responses to ANG II. These results suggest that the adrenergic neurotransmission in the MnPO may actively participate in ANG II-induced dipsogenesis, natriuresis, kaliuresis, diuresis and pressor responses in a process that involves α1-, α2-, and β-adrenoceptors.

Structural and kinetic studies of Schistosoma mansoni adenylate kinases

The human parasite Schistosoma mansoni is totally dependent on the purine salvage pathway in order to supply large quantities of purine precursors for its energy and DNA biosynthetic needs. Adenylate kinase (ADK) is responsible for the conversion of AMP (produced by the adenosine kinase reaction) into ADP, which is subsequently converted into ATP by nucleoside diphosphate kinase (NDPK). ADK and NDPK are the most active enzymes of the pathway, probably reflecting an evolutionary adaptation due to the intense use of the branch in which they participate. However, notwithstanding their importance very little information has been accumulated found regarding these enzymes. In this work two adenylate kinases from S. mansoni were cloned and heterologously expressed in Escherichia coil. The purified products were utilized in activity assays, and displayed kinetic parameters similar to the corresponding human orthologous proteins. The cytosolic S. mansoni ADK was crystallized and its structure solved allowing us to detect a difference in the nucleotide binding site when compared with the human ortholog. (C) 2012 Elsevier B.V. All rights reserved.

Hemolymph ion regulation and kinetic characteristics of the gill (Na+, K+)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na+, K+)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45 parts per thousand salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 +/- 22.1 mOsm kg(-1) H2O) at 45 parts per thousand but elevated compared to fresh-caught crabs (801.0 +/- 40.1 mOsm kg(-1) H2O). Hemolymph [Na+ (323.0 +/- 2.5 mmol L-1) and [Me2+) (34.6 +/- 1.0 mmol L-1) are hypo-regulated while [Ca2+] (22.5 +/- 0.7 mmol L-1) is hyper-regulated; [K+] is hyper-regulated in fresh-caught crabs (17.4 +/- 0.5 mmol L-1) but hypo-regulated (6.2 +/- 0.7 mmol L-1) at 45 parts per thousand. Protein expression patterns are altered in the 45 parts per thousand-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na+, K+)-ATPase alpha-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 +/- 3.5 U mg(-1); K-0.5 = 7.07 +/- 0.01 mu mol L-1) and a low-affinity ATP binding site (Vm = 108.1 +/- 2.5 U mg(-1); K-0.5 = 0.11 +/- 0.3 mmol L-1), both obeying cooperative kinetics, were disclosed. Modulation of (Na+, K+)-ATPase activity by Mg2+, K+ and NH4+ also exhibits site-site interactions, but modulation by Na+ shows Michaelis-Menten kinetics. (Na+, K+)-ATPase activity is synergistically stimulated up to 45% by NH4+ plus K+. Enzyme catalytic efficiency for variable [K+] and fixed [NH4+] is 10-fold greater than for variable [NH4+] and fixed [K+]. Ouabain inhibited approximate to 80% of total ATPase activity (K-I=464.7 +/- 23.2 mu mol L-1), suggesting that ATPases other than (Na+, K+)-ATPase are present. While (Na+, K+)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min(-1) mg(-1)) and 45 parts per thousand-acclimated crabs (around 154 nmol Pi min(-1) mg(-1)), ATP affinity decreases 110-fold and Na+ and K+ affinities increase 2-3-fold in 45 parts per thousand-acclimated crabs. (C) 2012 Elsevier Inc. All rights reserved.

The Integration of the Glutamatergic and the White Matter Hypotheses of Schizophrenia's Etiology

Background: schizophrenia's endophenotipic profile is not only generally complex, but often varies from case to case. The perspective of trying to define specific anatomic correlates of the syndrome has led to disappointing results. In that context, neurophysiologic hypotheses (e. g. glutamatergic hypothesis) and connectivity hypotheses became prominent. Nevertheless, despite their commitment to the principle of denying 'localist' views and approaching the syndrome's endophenotype from a whole brain perspective, efforts to integrate both have not flourished at this moment in time. Objectives: This paper aims to introduce a new etiological model that integrates the glutamatergic and the WM (WM) hypotheses of schizophrenia's etiology. This model proposes to serve as a framework in order to relate to patterns of brain abnormalities from the onset of the syndrome to stages of advanced chronification. Highlights: Neurotransmitter abnormalities forego noticeable WM abnormalities. The former, chiefly represented by NMDAR hypo-function and associated molecular cascades, is related to the first signs of cell loss. This process is both directly and indirectly integrated to the underpinning of WM structural abnormalities; not only is the excess of glutamate toxic to the WM, but its disruption is associated to the expression of known genetic risk factors (e. g., NRG-1). A second level of the model develops the idea that abnormal neurotransmission within specific neural populations ('motifs') impair particular cognitive abilities, while subsequent WM structural abnormalities impair the integration of brain functions and multimodality. As a result of this two-stage dynamic, the affected individual progresses from experiencing specific cognitive and psychological deficits, to a condition of cognitive and existential fragmentation, linked to hardly reversible decreases in psychosocial functioning.

Ionotropic Glutamate Receptors in Hypothalamic Paraventricular and Supraoptic Nuclei Mediate Vasopressin and Oxytocin Release in Unanesthetized Rats

We report changes in plasma arginine vasopressin (AVP) and oxytocin (OT) concentrations evoked by the microinjection of L-glutamate (L-glu) into the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus(PVN) of unanesthetized rats, as well as which local mechanisms are involved in their mediation. L-Glu microinjection (10 nmol/100 nl) into the SON increased the circulating levels of both AVP and OT. The AVP increases were blocked by local pretreatment with the selective non-N-methyl-D-aspartate (NMDA) receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (2 nmol/100 nl), but it was not affected by pretreatment with the NMDA-receptor antagonist LY235959 (2 nmol/100 nl). The OT response to L-glu microinjection into the SON was blocked by local pretreatment with either NBQX or LY235959. Furthermore, the administration of either the non-NMDA receptor agonist (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) (5 nmol/100 nl) or NMDA receptor agonist NMDA (5 nmol/100 nl) into the SON had no effect on OT baseline plasma levels, but when both agonists were microinjected together these levels were increased. L-Glu microinjection into the PVN did not change circulating levels of either AVP or OT. However, after local pretreatment with LY235959, the L-glu microinjection increased plasma levels of the hormones. The L-glu microinjection into the PVN after the local treatment with NBQX did not affect the circulating AVP and OT levels. Therefore, results suggest the AVP release from the SON is mediated by activation of non-NMDA glutamate receptors, whereas the OT release from this nucleus is mediated by an interaction of NMDA and non-NMDA receptors. The present study also suggests an inhibitory role for NMDA receptors in the PVN on the release of AVP and OT. (Endocrinology 153: 2323-2331, 2012)

Structural insights regarding an insecticidal Talisia esculenta protein and its biotechnological potential for Diatraea saccharalis larval control

Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinase-inhibitor properties. The present study aims to provide new in vitro and in silico biochemical information about this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensional structure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Since the structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried out using Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted non-coincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-type inhibitors, which demonstrate a substitution of an Arg(64) for the Glu(64) residue. Data, therefore, provide more information regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisin also presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstrating LD50 and ED50 of ca. 2.0% and 1.5%, respectively. (C) 2011 Elsevier Inc. All rights reserved.

Endothelium dependent expression and underlying mechanisms of des-Arg(9)-bradykinin-induced B1R-mediated vasoconstriction in rat portal vein

Endothelial dysfunction has been implicated in portal vein obstruction, a condition responsible for major complications in chronic portal hypertension. Increased vascular tone due to disruption of endothelial function has been associated with an imbalance in the equilibrium between endothelium-derived relaxing and contracting factors. Herein, we assessed underlying mechanisms by which expression of bradykinin B-1 receptor (B1R) is induced in the endothelium and how its stimulation triggers vasoconstriction in the rat portal vein. Prolonged in vitro incubation of portal vein resulted in time- and endothelium-dependent expression of B1R and cyclooxygenase-2 (COX-2). Inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3K) significantly reduced expression of B1R through the regulation of transcription factors, activator protein-1 (AP-1) and cAMP response element-binding protein (CREB). Moreover, pharmacological studies showed that B1R-mediated portal vein contraction was reduced by COX-2, but not COX-1, inhibitors. Notably, activation of endothelial B1R increased phospholipase A(2)/COX-2-derived thromboxane A(2) (TXA(2)) levels, which in turn mediated portal vein contraction through binding to TXA(2) receptors expressed in vascular smooth muscle cells. These results provide novel molecular mechanisms involved in the regulation of B1R expression and identify a critical role for the endothelial B1R in the modulation of portal vein vascular tone. Our study suggests a potential role for B1R antagonists as therapeutic tools for diseases where portal hypertension may be involved. (C) 2012 Elsevier Inc. All rights reserved.

Analysis of the NMDA in Focal Cerebral Ischemia in Rats

NMDAR (N-methyl-D-aspartate receptor) is one subtype of ionotrophic glutamate receptor which is extensively distributed in the central nervous system (CNS). In the mammalian CNS, NMDAR serves prominent roles in the pathophysiologic process of cerebral ischemia. This study aimed to investigate the pattern of expression of protein and gene of the excitatory neurotransmitter NMDAR in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. 120 rats were randomly divided into 6 groups (20 animals each): control - no surgery; sham - simulation of surgery; ischemic - focal ischemia for 1 hour, without reperfusion; ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. Ten animals from each experimental group were used to establish the volume of infarct. Transient focal cerebral ischemia was obtained in rats by occlusion of the middle cerebral artery with an intraluminal suture. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the protein and gene NMDA were evaluated by immunohistochemistry and quantitative real-time PCR, respectively. Increases in the protein and gene NMDA receptor in the ischemics areas were observed and these increases were reduced by hypothermia and ketoprofen. The increase in the NMDA receptor protein and gene expression observed in the ischemic animals was reduced by neuroprotection (hypothermia and ketoprofen). The NMDA receptor increases in the ischemic area suggests that the NMDA mediated neuroexcitotoxicity plays an important role in cell death and that the neuroprotective effect of both, hypothermia and ketoprofen is directly involved with the NMDA.

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913

GLUTAMATERGIC MECHANISMS OF THE DORSAL PERIAQUEDUCTAL GRAY MATTER MODULATE THE EXPRESSION OF CONDITIONED FREEZING AND FEAR-POTENTIATED STARTLE

It is well known that excitatory amino acids induce unconditioned fear responses when locally injected into the dorsal periaqueductal gray matter (dPAG). However, there are only few studies about the involvement of excitatory amino acids mediation in dPAG in the expression of conditioned fear. The present series of experiments evaluates the participation of AMPA/Kainate and NMDA glutamatergic receptors of dPAG in the expression of conditioned fear, assessed by the fear-potentiated startle (FPS) and conditioned freezing responses. Wistar rats were subjected to fear conditioning to light. Twenty-four hours later, they received intra-dPAG injections of kainic acid or NMDA (AMPA/Kainate and NMDA agonists) and 1,2,3,4-Tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium salt hydrate (NBQX) or D(-)-2-Amino-7-phosphonoheptanoic acid (APT) (AMPA/Kainate and NMDA antagonists) and were submitted to the FPS test. Conditioned freezing response was simultaneously measured. Effects of drug treatment on motor activity were evaluated in the open-field test. Intra-dPAG injections of glutamatergic agonists enhanced conditioned freezing and promoted pro-aversive effects in the FPS. Lower doses of the agonists had no effect or enhanced FPS whereas higher doses disrupted FPS, indicating a non-monotonic relationship between fear and FPS. The antagonist NBQX had no significant effects while AP7 decreased conditioned freezing but did not affect FPS. Both antagonists reduced the effects of the agonists. The obtained results cannot be attributed to motor deficits. The results suggest an important role of the AMPA/Kainate and NMDA mechanisms of the dPAG in the expression of conditioned freezing and FPS. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.