Detection of DNA Nucleotides on Pretreated Boron Doped Diamond Electrodes

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

Signaling path of the action of AVP on distal K+ secretion

Background. Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (J(K)) via V1 receptors (Am J Physiol 278: F809- F816, 2000). In the present work, we investigate the cell signaling mechanism of this process.Methods. In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K was measured using double K+ resin/reference microelectrodes.Results. In control conditions, J(K) was 0.71 +/- 0.05 nmol. cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, J(K) increased to 0.88 +/- 0.08 nmol. cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, J(K) was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca2+-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated J(K) by 36.4%, and AVP-independent J(K) (after blocking V1 receptors) by only 16%.Conclusion. The results suggest that the luminal V1-receptor effect of AVP on J(K) was mediated by the phospholipase C (PLC)/ Ca2+/PKC signaling path and not by adenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Ação do forskolin em diferentes fases da produção in vitro: implicação na meiose oocitária e na vitrificação de embriões bovinos

Pós-graduação em Medicina Veterinária - FMVZ

Interaction of Organophosphorus Pesticides with DNA Nucleotides on a Boron-Doped Diamond Electrode

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

Control of oocyte maturation

Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte.

Obesity does not lead to imbalance between myocardial phospholamban phosphorylation and dephosphorylation

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

Análise metabolômica do cérebro de abelhas (Apis mellifera) submetidas a ensaio de Reflexo de Extensão de Probóscide (REP)

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

Obtenção de amido resistente por intercruzamento e por tratamento hidrotérmico, e sua incorporação em bolos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

PTEN Directly Activates the Actin Depolymerization Factor Cofilin-1 During PGE(2)-Mediated Inhibition of Phagocytosis of Fungi

Macrophage ingestion of the yeast Candida albicans requires its recognition by multiple receptors and the activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E-2 (PGE(2)) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here, we characterized the mechanisms underlying PGE(2)-mediated inhibition of phagocytosis and filamentous actin (F-actin) polymerization in response to ingestion of C. albicans by alveolar macrophages. PGE(2) suppressed phagocytosis and F-actin formation through the PGE(2) receptors EP2 and EP4, cAMP, and activation of types I and II protein kinase A. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 were necessary for these inhibitory effects of PGE(2). PGE(2)-dependent activation of cofilin-1 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because enhanced production of PGE(2) accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

Abstract Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design. Results The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP) in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP. Conclusion Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

Changes in CREB activation in the prefrontal cortex and hippocampus blunt ethanol-induced behavioral sensitization in adolescent mice

Drug dependence is a major health problem in adults and has been recognized as a significant problem in adolescents. We previously demonstrated that repeated treatment with a behaviorally sensitizing dose of ethanol in adult mice induced tolerance or no sensitization in adolescents and that repeated ethanol-treated adolescents expressed lower Fos and Egr-1 expression than adult mice in the prefrontal cortex (PFC). In the present work, we investigated the effects of acute and repeated ethanol administration on cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) DNA-binding activity using the electrophoretic mobility shift assay (EMSA) and the phosphorylated CREB (pCREB)/CREB ratio using immunoblotting in both the PFC and hippocampus in adolescent and adult mice. Adult mice exhibited typical locomotor sensitization after 15 days of daily treatment with 2.0 g/kg ethanol, whereas adolescent mice did not exhibit sensitization. Overall, adolescent mice displayed lower CREB binding activity in the PFC compared with adult mice, whereas opposite effects were observed in the hippocampus. The present results indicate that ethanol exposure induces significant and differential neuroadaptive changes in CREB DNA-binding activity in the PFC and hippocampus in adolescent mice compared with adult mice. These differential molecular changes may contribute to the blunted ethanol-induced behavioral sensitization observed in adolescent mice.

Enhancement of behavioral sensitization, anxiety-like behavior, and hippocampal and frontal cortical CREB levels following cocaine abstinence in mice exposed to cocaine during adolescence

Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system

Mechanismen zur Suppression der experimentellen akuten Graft-versus-Host-Disease

Die Transplantation von allogenen hämatopoetischen Stammzellen stellt für viele Patienten mit hämatologischen Erkrankungen, wie beispielsweise akuter Leukämie, oftmals die einzige kurative Therapieoption dar. Die Erkennung von Empfängerantigenen durch immunkompetente Zellen des Spenders bietet dabei die Basis für erwünschte Graft-versus-Tumor-Effekte, verursacht jedoch häufig außerdem die unerwünschte Graft-versus-Host Disease (GvHD), eine mitunter schwerwiegende Komplikation. In der vorliegenden Arbeit wurden potentielle Mechanismen zur Hemmung alloreaktiver CD4+ und CD8+ T-Zellen (TZ) und folglich zur Hemmung der akuten GvHD in einem experimentellen GvHD-Modell untersucht, welches auf dem Transfer von allogenen Zellen zwischen MHC-inkompatiblen Mausstämmen basiert. Die vorliegende Arbeit weist zum Einen darauf hin, dass das Fehlen MyD88- und TRIF-vermittelter Toll-like-Rezeptor-Signale zumindest im Rahmen des hier verwendeten Transplantationsmodells nicht zwingend zu einer Hemmung der akuten GvHD führt. Zum Anderen konnte belegt werden, dass CD4+ CD25+ regulatorische T-Zellen (Tregs) kompetente Suppressoren der durch alloreaktive CD4+ und CD8+ TZ ausgelösten akuten GvHD darstellen. In weiterführenden Experimenten ist gezeigt worden, dass die Tregs sich verschiedener Mechanismen bedienen, um ihre Zielzellen zu inhibieren. Das suppressive Zytokin Interleukin-10 kann als löslicher Mediator zumindest in vitro offenbar eine Rolle bei der Treg-vermittelten Suppression alloreaktiver TZ spielen. Da jedoch auch Tregs aus Interleukin-10-defizienten Spendern die GvHD-Entstehung in den Empfängern abschwächen konnten, müssen noch weitere Mechanismen involviert sein. Es konnte in einer gemischten Leukozyten Reaktion in vitro eine zellkontaktabhängige Kommunikation mittels gap junctions hauptsächlich zwischen den Tregs und den allogenen Dendritischen Zellen (DCs) nachgewiesen werden, welche prinzipiell den Transfer von cAMP möglich macht. Die Kommunikation zwischen Tregs und DCs resultierte in einem supprimierten Phänotyp der DCs, gekennzeichnet durch eine verminderte Expression kostimulatorischer Moleküle auf ihrer Oberfläche. Solche supprimierten DCs können als Folge die alloreaktiven Spender-TZ vermutlich nicht aktivieren. Das cAMP-erhöhende Rolipram konnte in einer gemischten Leukozyten Reaktion in vitro die Proliferation alloreaktiver CD4+ und CD8+ TZ hemmen. Daneben konnte die Treg-vermittelte Suppression alloreaktiver TZ und der GvHD in vivo durch die zusätzliche Verabreichung von Rolipram noch gesteigert werden. Im letzten Kapitel dieser Arbeit wurde beschrieben, dass die alleinige Aktivierung alloreaktiver CD8+ TZ ausreichend ist, um eine akute GvHD auszulösen. In diesem Zusammenhang konnte nachgewiesen werden, dass CD4+ CD25+ Tregs die akute GvHD auch in einer scheinbar MHC-II-unabhängigen Weise hemmen können. Zusammenfassend belegt die vorliegende Arbeit, dass Tregs in einem MHC-inkompatiblen Transplantationsmodell alloreaktive CD4+ und CD8+ TZ und folglich die Entstehung einer GvHD effizient hemmen können. Bei der Hemmung der GvHD kommen wahrscheinlich verschiedene Mechanismen zum Tragen. Zumindest in vivo scheint von Tregs produziertes Interleukin-10 eine untergeordnete Rolle bei der Suppression alloreaktiver TZ und der GvHD zu spielen, hierbei steht vermutlich vielmehr der cAMP-abhängige Suppressionsmechanismus im Vordergrund.

Suppression des allergischen Asthmas durch regulatorische T-Zellen: Mechanismen und potenzielle therapeutische Ansätze

Das allergische Asthma ist eine weit verbreitete, immunologische Erkrankung, deren Prävalenz in den vergangenen 20 Jahren vor allem in industrialisierten Regionen drastisch zugenommen hat. Trotz intensiver Forschung und Entwicklung medikamentöser Therapien steigt die Zahl der Patienten stetig an. Charakteristisch für diese Erkrankung sind entzündliche Veränderungen in der Lunge, erhöhte Atemwegsüberempfindlichkeit (AHR), Mukusproduktion und in chronischen Fällen auch Atemwegsobstruktion. Bei der Entstehung des allergischen Asthmas wird ein anfälliges Individuum durch die Inhalation eines normalerweise unschädlichen, in der Umwelt vorkommenden Antigens (Allergen) sensibilisiert, wodurch im Körper eine eigentlich unangebrachte Immunreaktion in Gang gesetzt wird. CD4+ T-Lymphozyten und ganz besonders die Subpopulationen der T-Helfer 1 (Th1) und Th2 Zellen spielen in dem Prozess eine zentrale Rolle. Obwohl ein Großteil der Asthmatiker mit einer Atemwegseosinophilie und erhöhter Expression der Th2-typischen Zytokine IL-4 und IL-13 ein Th2-typisches Krankheitsbild aufweisen, wurden weitere Asthmaphänotypen identifiziert. Vornehmlich in Patienten, die an schwerem Asthma leiden, sind dominierende Neutrophilie und erhöhte Mengen IFN-γ in den Atemwegen nachweisbar, was auf eine Th1-gesteuerte Immunreaktion hindeutet. Eine effektive, heilende Therapie des Asthmas wurde bislang nicht entwickelt. Die Inhibition der T-Zellantwort etwa durch Applikation allergenspezifischer, regulatorischer T-Zellen (Tregs) gilt als ein vielversprechender, aber nicht vollständig erforschter Ansatz zur Kontrolle der Krankheitssymptome. In diesem Zusammenhang wurden in der vorliegenden Arbeit die Mechanismen und Effekte natürlich vorkommender CD4+CD25+Foxp3+ regulatorischer T-Zellen (nTregs) auf eine Th1 bzw. Th2-induzierte allergische Atemwegserkrankung untersucht. Anhand eines adoptiven Zelltransfermodells unter Einsatz lymphozytendefizienter Rag2-/- Mäuse konnte gezeigt werden, dass sowohl Th1 als auch Th2 Zellen, kombiniert mit mehrfacher, inhalativer Allergenprovokation, eine erhöhte AHR induzieren. Während der Transfer allergenspezifischer Th2 Zellen eine Eosinophilie in der bronchoalveolären Lavage (BAL) und vermehrte Mukusproduktion in den Atemwegen hervorrief, war in Th1-transferierten Tieren zwar eine massive Infiltration neutrophiler Granulozyten zu beobachten, eine Becherzellmetaplasie mit vermehrten, mukusproduzierenden Atemwegsepithelzellen blieb allerdings aus. In vitro und in vivo waren voraktivierte nTregs (preTregs) nur eingeschränkt in der Lage, die Th2-gesteuerte Atemwegserkrankung zu inhibieren. Im Gegensatz dazu konnten die Th1-Effektorfunktionen in vitro und die Th1-induzierte AHR und Atemwegsentzündung in vivo durch preTregs effektiv gehemmt werden, was auf eine unterschiedliche Empfindlichkeit der Th-Subpopulationen weist. Innerhalb der nTreg-vermittelten Suppression wird der sekundäre Botenstoff cAMP auf die zu supprimierende Zelle übertragen und führt zur Hemmung von Proliferation und Zytokinproduktion. Dass dieser Mechanismus nicht nur in vitro, sondern auch in der Suppression der Th2-gesteuerten allergischen Atemwegserkrankung eine Rolle spielt, konnte durch die Störung des intrazellulären cAMP-Abbaus mittels PDE4-Inhibitoren verdeutlicht werden. Sowohl die prophylaktische, als auch die therapeutische Applikation der PDE4-Inhibitoren verstärkte den regulativen Effekt der nTregs auf AHR und Entzündung, korrelierend mit erhöhten, zytosolischen cAMP-Konzentrationen in den Th2 Zellen der Lunge. Trotz des Fortschritts in der Isolation und In vitro-Expansion humaner nTregs ist die Ausbeute an Zellen äußerst limitiert und die Übertragbarkeit größerer Zellmengen nicht zuletzt aufgrund von hohem Kontaminationsrisiko während mehrtägiger In vitro-Expansion fragwürdig. Die Ergebnisse der vorliegenden Arbeit zeigen, dass eine Behandlung mit dem PDE4-Inhibitor die suppressive Kapazität der allergenspezifischen nTregs deutlich erhöhte. Den nTreg-vermittelten Suppressionsmechanismus durch den Einsatz von Pharmazeutika zu unterstützen bietet einen viel versprechenden und realistischen Ansatz zur Therapie des allergischen Asthmas.