A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1 beta/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1 beta and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

The zymogen-enteropeptidase system: A practical approach to study the regulation of enzyme activity by proteolytic cleavage

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly switched on by specific proteolytic cleavage.

GABA(A) receptor activation in the lateral parabrachial nucleus induces water and hypertonic NaCl intake

Inhibitory serotonergic and cholecystokinergic mechanisms in the lateral parabrachial nucleus and central GABAergic mechanisms are involved in the regulation of water and NaCl intake. In the present study we investigated if the GABA(A) receptors in the lateral parabrachial nucleus are involved in the control of water, NaCl and food intake in rats. Male Holtzman rats with stainless steel cannulas implanted bilaterally into the lateral parabrachial nucleus were used. Bilateral injections of muscimol (0.2 nmol/0.2 mu l) into the lateral parabrachial nucleus strongly increased 0.3 M NaCl (20.3 +/- 7.2 vs. saline: 2.6 +/- 0.9 ml/180 min) without changing water intake induced by the treatment with the diuretic furosemide combined with low dose of the angiotensin converting enzyme inhibitor captopril s.c. In euhydrated and satiated rats, bilateral lateral parabrachial nucleus injections of muscimol (0.2 and 0.5 nmol/0.2 0) induced 0.3 M NaCl intake (12.1 +/- 6.5 and 32.5 +/- 7.3 ml/180 min, respectively, vs. saline: 0.4 +/- 0.2 ml/180 min) and water intake (5.2 +/- 2.0 and 7.6 +/- 2.8 ml/ 180 min, respectively, vs. saline: 0.8 +/- 0.4 ml/180 min), but no food intake (2 +/- 0.4 g/240 min vs. saline: 1 +/- 0.3 g/240 min). Bilateral lateral parabrachial nucleus injections of the GABAA antagonist bicuculline (1.6 nmol/0.2 mu l) abolished the effects of muscimol (0.5 nmol/0.2 mu l) on 0.3 M NaCl and water intake. Muscimol (0.5 nmol/0.2 mu l) into the lateral parabrachial nucleus also induced a slight ingestion of water (4.2 +/- 1.6 ml/240 min vs. saline: 1.1 +/- 0.3 ml/240 min) when only water was available, a long lasting (for at least 2 h) increase on mean arterial pressure (14 +/- 4 mm Hg, vs. saline: -1 +/- 1 mm Hg) and only a tendency to increase urinary volume and Na+ and K+ renal excretion. Therefore the activation of GABAA receptors in the lateral parabrachial nucleus induces strong NaCl intake, a small ingestion of water and pressor responses, without changes on food intake. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

Activation of serotonergic 5-HT1A receptors in the lateral parabrachial nucleus increases NaCl intake

Previous studies using non-specific serotonergic agonists and antagonists have shown the importance of serotonergic inhibitory mechanisms in the lateral parabrachial nucleus (LPBN) for controlling sodium and water intake. In the present study, we investigated whether the serotonergic 5-HTIA receptor subtype in the LPBN participates in this control. Male Holtzman rats had cannulas implanted bilaterally into the LPBN. Bilateral injections of the 5-HTIA receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.1, 1.25, and 2.5 mu g/ 0.2 mu l), into the LPBN enhanced 0.3 M NaCl and water intake of rats injected subcutaneously with the diuretic furosemide (10 mg/kg bw) and a low dose of the angiotensin-converting enzyme inhibitor, captopril (5 mg/kg bw). The increase in NaCl intake produced by 8-OH-DPAT injections was reduced in dose-related manner by pre-treating the LPBN with the selective 5-HTIA serotonergic antagonist, WAY-100635 (WAY, I and 2 mu g/0.2 mu l). In contrast, WAY did not affect water intake produced by 8-OH-DPAT. WAY-100635 injected alone into the LPBN had no effect on NaCl ingestion. Injections of 8-OH-DAPT (0.1 mu g/0.2 mu l) into the LPBN also increased 0.3 M NaCl intake induced by 24-h sodium depletion (furosemide, 20 mg/kg bw plus 24 h of sodium-free diet). Serotonin (5-HT, 20 mu g/0.2 mu l) injected alone or combined with 8-OH-DPAT into the LPBN reduced 24-h sodium depletion-induced 0.3 M NaCl intake. Therefore, the activation of serotonergic 5-HTIA receptors in the LPBN increases stimulated hypertonic NaCl and water intake, while 5-HT injections into the LPBN reduce NaCl intake and prevent the effects of serotonergic 5-HTIA receptor activation. (c) 2005 Elsevier B.V. All rights reserved.

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases - Crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 angstrom resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42-.

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A(2) promoted by amphiphilic molecules

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca2+-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca2+ ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)S), substitution of the Asp49 by Lys results in loss of Ca2+ binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA2S cause Ca2+-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca2+-independent membrane damaging activity by similar to4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accomparlied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s. (C) 2004 Published by Elsevier B.V.

Influence of activation on the multipoint immobilization of penicillin G acylase on macroporous silica

Penicillin G acylase is the second most important enzyme used by industry in an immobilized form. Penicillin hydrolysis is its main application. This reaction is used to produce 6-aminopenicillanic acid (6-APA), an intermediate in the synthesis of semisynthetic antibiotics. This work aims to compare catalytic properties of different penicillin G acylase (PGA) derivatives obtained by multipoint immobilization of the enzyme on macroporous silica. Enzyme amino groups react with different aldehyde groups produced in the support using either glutaraldehyde or glyoxyl activation. In the former method, silica reacts with g-aminopropyltriethoxysilane (g-APTS) and glutaraldehyde; in the latter, a reaction with glycidoxypropyltrimethoxysilane (GPTMS) is followed by acid hydrolysis and oxidation using sodium periodate. This work determines the influence of degree of activation, using glutaraldehyde, on immobilization parameters. PGA was immobilized on these two different supports. Maximum enzyme load, immobilized enzyme activity (derivative activity), rate of immobilization and thermal stability were checked for both cases. For glutaraldehyde activation, the results showed that 0.5% of the g-APTS is sufficient for all the hydroxyl groups in the silica to react. They also showed that degree of activation only affects immobilization yield and reaction velocity and that reduction of the glutaraldehyde derivatives with sodium borohydride does not affect their thermal stability. In comparing the derivatives obtained using glyoxyl and glutaraldehyde activation, it was observed that the glyoxyl derivatives presented better immobilization parameters, with a maximum enzyme load of 264 IU/g silica and a half-life of 20 minutes at 60 °C.

CENTRAL ANGIOTENSIN-CONVERTING ENZYME-BLOCKADE AND THIRST

1. The effect of lisinopril, a potent inhibitor of angiotensin converting enzyme (ACE), injected into the medial preoptic area (MPOA) on water intake was investigated in male Holtzman rats (200-250 g).2. Injection of lisinopril (2 mug/mul) into the MPOA abolished the water intake induced by subcutaneous (sc) injection of isoprenaline (100%) and water deprivation (90%) and drastically reduced the water intake induced by sc injection of polyethyleneglycol (60%). A small reduction of water intake induced by lisinopril was also observed 90 and 120 min after sc hypertonic saline (N = 10 for each group).3. These results suggest that central ACE activation, particularly in the MPOA, plays an important role in the dipsogenic responses induced by the agents studied.

Anxiogenic-like effects induced by NMDA receptor activation are prevented by inhibition of neuronal nitric oxide synthase in the periaqueductal gray in mice

Glutamate-NMDA (N-methyl-D-aspartate) receptor activation within the periaqueductal gray (PAG) leads to antinociceptive, autonomic and behavioral responses characterized as the fear reaction. We have recently demonstrated that the vigorous defensive-like behaviors (e.g. jumping and running) and antinociception induced by intra-PAG injection of N-methyl-D-aspartate (NMDA) were completely blocked by prior infusion of N(omega)-propyl-L-arginine (NPLA), a specific neuronal nitric oxide synthesis (nNOS) enzyme inhibitor, into the same midbrain structure. It remains unclear however, whether the inhibition of nNOS within the mouse PAG changes the anxiety-like behavior per se or the effects of the inhibition of nNOS depend on the suppression of downstream of glutamate-NMDA receptor activation. This study investigated whether intra-PAG infusion of NPLA (i) attenuates anxiety in the elevated plus-maze (EPM) and (ii) antagonizes the anxiogenic-like effects induced by intra-PAG injection of NMDA. Test sessions were videotaped and subsequently scored for conventional indices of anxiety (percentage of open arm entries and percentage of open arm time) and locomotor activity (closed arm entries). Results showed that intra-PAG infusions of NPLA (0.2, 0.4 or 0.8 nmol/0.1 mu l) did not alter significantly any behavioral response in the EPM when compared to control group (Experiment 1). Intra-PAG infusion of NMDA (0 and 0.02 nmol/0.1 mu l; a dose that does not provoke vigorous defensive behaviors per se in mice) significantly reduced open arm exploration, confirming an anxiogenic-like effect (Experiment 2). When injected into the PAG 10 min prior local NMDA injection (0.02 nmol/0.1 mu l), NPLA (0.4 nmol/0.1 mu l) was able to revert the anxiogenic-like effect of glutamate-NMDA receptor activation. Neither intra-PAG infusion of NMDA nor NPLA altered closed arm entries, a widely used measure of locomotor activity in the EPM. These results suggest that intra-PAG nitric oxide synthesis does not play a role on anxiety-like behavior elicited during EPM exposure; however its synthesis is important for the proaversive effects produced by activation of glutamate-NMDA receptors located within this limbic midbrain structure. (C) 2008 Elsevier B.V. All rights reserved.

A new application of humic substances: Activation of supports for invertase immobilization

Invertase was immobilized on aminopropyl silica (APTS-SiO2) activated with humic substances (APTS-SiO2-HS) and on aminopropyl silica activated with glutaraldehyde (APTS-SiO2-GA). The resulting activity of both systems was compared. Humic substances (HS) used for the activation of the silica were extracted from soil of Cananéia, São Paulo State, Brazil, according to the procedure recommended by the International Humic Substances Society. Activity was determined by measuring the rate of formation of reduced sugars using the reaction with dinitrosalicylic acid (DNS). The amount of HS bound on the APTS-SiO2 was equal to 50 mg. The maximum amount of invertase immobilized on APTS-SiO2-HS was 15200 U/g while in the system APTS-SiO2-GA it was 13400 U/g. The experimental enzymatic activity was 3700 and 3300 U/g, for the systems APTS-SiO2-HS and APTS-SiO2-GA, respectively. Considering the increased amount and activity of immobilized enzyme compared with the glutaraldehyde method, it was concluded that this technique opens a new perspective in the preparation of supports for enzyme immobilization employing humic substances. © Springer-Verlag 2000.

Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFβ-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-γ and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-β1 and IL-4 production by BALB/c mice and to an increase in the IFN-γ levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.

Involvement of central cholinergic mechanisms on sodium intake induced by gabaergic activation of the lateral parabrachial nucleus

Bilateral injections of the GABAA agonist muscimol into the lateral parabrachial nucleus (LPBN) disrupt satiety and induce strong ingestion of water and 0.3M NaCl in fluid-replete rats by mechanisms not completely clear. In the present study, we investigated the effects of the blockade of central muscarinic cholinergic receptors with atropine injected intracerebroventricularly (i.c.v.) on 0.3M NaCl and water intake induced by muscimol injections into the LPBN in fluid-replete rats. Male Holtzman rats with stainless steel cannulas implanted bilaterally into the LPBN and unilaterally into the lateral ventricle (LV) were used. Bilateral injections of muscimol (0.5nmol/0.2μL) into the LPBN induced 0.3M NaCl (32.2±9.9mL/4h, vs. saline: 0.4±0.2mL/4h) and water intake (11.4±4.4mL/4h, vs. saline: 0.8±0.4mL/4h) in fluid-replete rats previously treated with i.c.v. injection of saline. The previous i.c.v. injection of atropine (20nmol/1μL) reduced the effects of LPBN-muscimol on 0.3M NaCl (13.5±5.0mL/4h) and water intake (2.9±1.6mL/4h). The i.c.v. injection of atropine did not affect 0.3M NaCl (26.8±6.2mL/2h, vs. saline i.c.v.: 36.5±9.8mL/2h) or water intake (14.4±2.5mL/2h, vs. saline i.c.v.: 15.6±4.8mL/2h) in rats treated with furosemide+captopril subcutaneously combined with bilateral injections of moxonidine (α2-adrenoceptor/imidazoline agonist, 0.5nmol/0.2μL) into the LPBN, suggesting that the effect of atropine was not due to non-specific inhibition of ingestive behaviors. The results show that active central cholinergic mechanisms are necessary for the hypertonic NaCl and water intake induced by the blockade of the inhibitory mechanisms with injections of muscimol into the LPBN in fluid-replete rats. The suggestion is that in fluid-replete rats the action of LPBN mechanisms inhibits facilitatory signals produced by the activity of central cholinergic mechanisms to maintain satiety. © 2012 Elsevier B.V.

Signaling Pathways Activation by Primary Endodontic Infectious Contents and Production of Inflammatory Mediators

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

Macrophage Cell Activation with Acute Apical Abscess Contents Determined by Interleukin-1 Beta and Tumor Necrosis Factor Alpha Production

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