Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 mu g/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX-blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Argon ion laser and halogen lamp activation of a dark and light resin composite: microhardness after long-term storage

The objective of this study was to evaluate in vitro light activation of the nano-filled resin composite Vita shade A1 and A3 with a halogen lamp (QTH) and argon ion laser by Knoop microhardness profile. Materials and methods: Specimens of nanofilled composite resin (Z350-3 M-ESPE) Vita shade A1 and A3 were prepared with a single increment inserted in 2.0-mm-thick and 3-mm diameter disc-shaped Teflon mold. The light activation was performed with QTH for 20 s (with an intensity of approximately 1,000 mW/cm(2) and 700 mW/cm(2)) and argon ion laser for 10 s (with a power of 150 mW and 200 mW). Knoop microhardness test was performed after 24 h and 6 months. The specimens were divided into the 16 experimental groups (n = 10), according to the factors under study: photoactivation form, resin shade, and storage time. Knoop microhardness data was analyzed by a factorial ANOVA and TukeyA ` s tests at the 0.05 level of significance. Results: Argon ion laser was not able to photo-activate the darker shade of the nanofilled resin composite evaluated but when used with 200 mW it can be as effective as QTH to photo-activate the lighter shade with only 50% of the time exposure. After 6 months storage, an increase in the means of Knoop microhardness values were observed. Conclusions: Light-activation significantly influenced the Knoop microhardness values for the darker nanofilled resin composite.

Qualitative Analysis of the Removal of the Smear Layer in the Apical Third of Curved Roots: Conventional Irrigation versus Activation Systems

Introduction: The aim of this study was to evaluate the effectiveness of different irrigant agitation techniques on smear layer removal in curved root canals. Methods: Mesiobuccal canals of 62 extracted lower molars with a curvature of 33 degrees were used and instrumented up to Pro Taper F2. The samples were divided into 3 experimental groups according to the final irrigation: conventional irrigation, ultrasonic irrigation, and sonic irrigation by using the Endo Activator system. The control group was composed of 2 specimens without any final irrigation. In all of the experimental groups, 5 mL of 17% ethylenediaminetetraacetic acid was used for 1 minute, and 5 mL of 2.5% NaOCl was used for 30 seconds. The analysis of the apical region was performed via scanning electron microscopy by 3 examiners. The data were submitted to the Kruskal-Wallis and Dunn tests (P<.05). Results: The activation systems removed significantly more smear layer than did conventional irrigation. Conclusions: Sonic and ultrasonic irrigation resulted in better removal of the smear layer in the apical third of curved root canals than did conventional irrigation. (J Endod 2011;37:1268-1271)

GABAergic antagonist blocks the reduction of tonic immobility behavior induced by activation of 5-HT(2) receptors in the basolateral nucleus of the amygdala in guinea pigs

Tonic immobility (TI) is a temporary state of profound motor inhibition induced by situations that generate intense fear, with the objective of protecting an animal from attacks by predators. A preliminary study by our group demonstrated that microinjection into the basolateral nucleus of the amygdala (BLA) of an agonist to 5-HT(1A) and 5-HT(2) receptors promoted a decrease in TI duration. In the current study, the effects of GABAergic stimulation of the BLA and the possible interaction between GABA(A) and 5-HT(2) receptors on TI modulation were investigated. Observation revealed that GABAergic agonist muscimol (0.26 nmol) reduced the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol) increased TI duration. Additionally, microinjection of 5-HT(2) agonist receptors (alpha-methyl-5-HT, 0.32 nmol) into the BLA decreased TI duration, an effect reversed by pretreatment with bicuculline (at the dose that had no effect per se, 0.2 nmol). Moreover, the activation of GABA(A) and 5-HT(2) receptors in the BLA did not alter the spontaneous motor activity in the open field test. These experiments demonstrated that the activation of GABA(A) and 5-HT(2) receptors of the BLA possibly produce a reduction in unconditioned fear that decreases the TI duration in guinea pigs, but this is not due to increased spontaneous motor activity, which could affect a TI episode nonspecifically. Furthermore, these results suggest an interaction between GABAergic and serotoninergic mechanisms mediated by GABA(A) and 5-HT(2) receptors. In addition, the GABAergic circuit of the BLA presents a tonic inhibitory influence on TI duration. (C) 2009 Elsevier Inc. All rights reserved.

Effects of short-term acetaminophen and celecoxib treatment on orthodontic tooth movement and neuronal activation in rat

Non-steroidal anti-inflammatory drugs (NSAIDs) have been used for pain relief in orthodontics, but clinical studies reported that they may reduce tooth movement (TM). By other side, TM seems to activate brain structures related to nociception, but the effects of NSAIDs in this activation have not been studied yet. We analyzed the effect of short-term treatment with acetaminophen or celecoxib in the separation of rat upper incisors, as well as in neuronal activation of the spinal trigeminal nucleus, following tooth movement. Thirty rats (400-420 g) were pretreated through oral gavage (1 ml/dose)with acetaminophen (200 mg/kg), celecoxib (50 mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance for TM. In controls, this appliance was immediately removed after its introduction. Rats received ground food, and every 12 h, one of the drugs or vehicle. After 48 h, they were anesthetized, maxilla was radiographed, and were perfused with 4% paraformaldehyde. Brains were further processed for Fos immunohistochemistry. TM induced incisor distalization (p < 0.05) and neuronal activation of the spinal trigeminal nucleus. Treatment with both drugs did not affect tooth movement, but reduced c-fos expression in the caudalis subnucleus. No changes in c-fos expression were seen in the oralis and interpolaris subnuclei. We conclude that neither celecoxib nor acetaminophen seems to affect tooth movement, when used for 2 days, but both drugs are able to reduce the activation of brain structures related to nociception. Short-term treatment with celecoxib, thus, may be a therapeutic alternative to acetaminophen when the latter is contra indicated. (C) 2009 Elsevier Inc. All rights reserved.

Blocking systemic nitric oxide production alters neuronal activation in brain structures involved in cardiovascular regulation during polymicrobial sepsis

In a previous study, we concluded that overproduction of nitric oxide (NO) by inducible nitric Oxide synthase (iNOS) in the late phase of sepsis prevents hypothalamic activation, blunts vasopressin secretion and contributes to hypotension, irreversible shock and death. The aim of this follow-up study was to evaluate if the same neuronal activation pattern happens in brain structures related to cardiovascular functions. Male Wistar rats received intraperitoneal injections of aminoguanidine, an iNOS inhibitor, or saline 30 min before cecal ligation and puncture (CLP) or sham surgeries. The animals were perfused 6 or 24 h after the surgeries and the brains were removed and processed for Fos immunocytochemistry We observed an increase (P < 0.001) in c-fos expression 6 h after CLP in the area postrema (AP), nucleus of he tractus solitarius (NTS), ventral lateral medulla (VLM), locus coeruleus (LC) and parabrachial nucleus (PB). At 24 h after CLP, however, c-fos expression was strongly decreased in all these nuclei (P < 0.05), except for the VLM. Aminoguanidine reduced c-fos expression in the AP and NTS at 6 h after CLR but showed an opposite effect at 24 h, with an increase in the AP, NTS, and also in the VLM. No such effect was observed in the LC and PB at 6 or 24 h. In all control animals, c-fos expression was minimal or absent. We conclude that in the early phase of sepsis iNOS-derived NO may be partially responsible for the activation of brain structures related to cardiovascular regulation. During the late phase, however, this activation is reduced or abolished. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Rapid maxillary expansion causes neuronal activation in brain structures of rats

A correlation between pain sensation and neuronal c-fos expression has been analyzed following experimental rapid maxillar expansion (RME). Adult male Wistar rats were anaesthetized and divided into three groups: animals that received an orthodontic apparatus, which was immediately removed after the insertion (control), animals that received an inactivated orthodontic apparatus (without force), and animals that received an orthodontic apparatus previously activated (140 g force). After 6, 24, 48, or 72 h, the animals were re-anaesthetized, and perfused with 4% paraformaldehyde. The brains were removed, fixed, and sections containing brain structures related to nociception were processed for Fos protein immunohistochemistry (IHC). The insertion of the orthodontic apparatus with 140 g was able to cause RME that could be seen by radiography. The IHC results showed that the number of activated neurons in the different nuclei changed according to the duration of appliance insertion and followed a temporal pattern similar to that of sensations described in clinics. The animals that received the orthodontic apparatus without force did not show RME but a smaller c-fos expression in the same brain structures. In conclusion, we demonstrate that orthodontic force used for palate disjunction activates brain structures that are related to nociception, and that this activation is related to the pain sensation described during orthodontic treatment. (c) 2008 Elsevier Inc. All rights reserved.

Myeloperoxidase activity is increased in gingival crevicular fluid and whole saliva after fixed orthodontic appliance activation

Introduction: Orthodontic tooth movement uses mechanical forces that result in inflammation in the first days. Myeloperoxidase (MPO) is an enzyme found in polymorphonuclear neutrophil (PMN) granules, and it is used to estimate the number of PMN granules in tissues. So far, MPO has not been used to study the inflammatory alterations after the application of orthodontic tooth movement forces. The aim of this study was to determine MPO activity in the gingival crevicular fluid (GCF) and saliva (whole stimulated saliva) of orthodontic patients at different time points after fixed appliance activation. Methods: MPO was determined in the GCF and collected by means of periopaper from the saliva of 14 patients with orthodontic fixed appliances. GCF and saliva samples were collected at baseline, 2 hours, and 7 and 14 days after application of the orthodontic force. Results: Mean MPO activity was increased in both the GCF and saliva of orthodontic patients at 2 hours after appliance activation (P<0.02 for all comparisons). At 2 hours, PMN infiltration into the periodontal ligament from the orthodontic force probably results in the increased MPO level observed at this time point. Conclusions: MPO might be a good marker to assess inflammation in orthodontic movement; it deserves further studies in orthodontic therapy. (Am J Orthod Dentofacial Orthop 2010;138:613-6)

Effects of calcining conditions on the microstructure of sugar cane waste ashes (SCWA): Influence in the pozzolanic activation

in this paper a study of calcining conditions on the microstructural features of sugar cane waste ash (SCWA) is carried out. For this purpose, some microparticles (< 90 mu m) of sugar cane straw ash and sugar cane bagasse ash of samples calcined at 800 degrees C and 1000 are studied by combining the bright field and the dark field images with the electron diffraction patterns in the transmission electron microscopy (TEM). It is appreciated that the morphology and texture of these microparticles change when silicon or calcium are present. Furthermore, it is observed that iron oxide (magnetite Fe(3)O(4)) is located in the calcium-rich particles. The microstructural information is correlated with the results of a kinetic-diffusive model that allows the computing of the kinetic parameters of the pozzolanic reaction (mainly the reaction rate constant). The results show that the sugar cane wastes ash calcined at 800 and 1000 degrees C have properties indicative of high pozzolanic activity. The X-ray diffraction patterns, the TEM images and the pozzolanic activity tests show the influence of different factors on the activation of these ashes. (c) 2008 Elsevier Ltd. All rights reserved.

Activation of Leishmania (Leishmania) amazonensis arginase at low temperature by binuclear Mn(2+) center formation of the immobilized enzyme on a Ni(2+) resin

In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn(2+) applied to the nickel column at 23 degrees C. The intensity of the binding of the enzyme to the Ni(2+) resin was directly proportional to the concentration of Mn(2+). Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni(2+), allowing the following to occur: (1) entrance of Mn(2+) and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 degrees C; and (3) an increase in the affinity of the enzyme to Ni(2+) after the Mn(2+) activation step. The conformational alterations can be summarized as follows: the interaction with the Ni(2+) simulates thermal heating in the artificial activation by opening a channel for Mn(2+) to enter. (C) 2010 Elsevier Inc. All rights reserved.

Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins

The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NF kappa B and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NF kappa B and AP-1. Activation of NF kappa B and AP-1 depended on phospholipase C and protein kinase A. Activation of NF kappa B also depended on protein kinase C. Isoprenaline activated both NF kappa B and AP-1, and phenylephrine activated NF kappa B and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Investigation of whole blood of SJL/J mice using neutron activation analysis

The Br (0.0022 +/- A 0.0006 gL(-1)), Ca (0.113 +/- A 0.012 gL(-1)), Cl (3.07 +/- A 0.36 gL(-1)), K (2.63 +/- A 0.14 gL(-1)), Mg (0.045 +/- A 0.002 gL(-1)) and Na (2.09 +/- A 0.10 gL(-1)) concentrations were determined in whole blood of SJL/J mice using the Neutron Activation Analysis (NAA) technique. Eleven whole blood samples were analyzed in the IEA-R1 nuclear reactor at IPEN (So Paulo, Brazil). These data contribute for applications in veterinary medicine related to biochemistry analyses using whole blood. Moreover, the correlation with human blood estimation allows to checking the similarities for studying muscular dystrophy using this model animal.

Acquisition of Pavlovian Fear Conditioning Using beta-Adrenoceptor Activation of the Dorsal Premammillary Nucleus as an Unconditioned Stimulus to Mimic Live Predator-Threat Exposure

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; beta-adrenoceptor agonist), with amyl acetate odor-the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of beta-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (beta-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd beta-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning. Neuropsychopharmacology (2011) 36, 926-939; doi:10.1038/npp.2010.231; published online 5 January 2011