Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital Microscopic Studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression Of L-selectin and beta(2)-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to NO, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. (C) 2009 Elsevier Inc. All rights reserved.

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.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. 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.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Prolonged consumption of soy or fish-oil-enriched diets differentially affects the pattern of hypothalamic neuronal activation induced by refeeding in rats

We used c-Fos immunoreactivity to estimate neuronal activation in hypothalamic feeding-regulatory areas of 3-month-old rats fed control or oil-enriched diets (soy or fish) since weaning. While no diet effect was observed in c-Fos immunoreactivity of 24-h fasted animals, the acute response to refeeding was modified by both hyperlipidic diets but with different patterns. Upon refeeding, control-diet rats had significantly increased c-Fos immunoreactivity only in the paraventricular hypothalamic nucleus (PVH, 142%). In soy-diet rats, refeeding with the soy diet increased c-Fos immunoreactivity in dorsomedial hypothalamic nucleus (DMH, 271%) and lateral hypothalamic area (LH, 303%). Refeeding fish-diet rats with the fish diet increased c-Fos immunoreactivity in PVH (161%), DMH (177%), VMH (81%), and ARC (127%). Compared to the fish-diet, c-Fos immunoreactivity was increased in LH by the soy-diet while it was decreased in ventromedial hypothalamic nucleus (VMH) and arcuate hypothalamic nucleus (ARC). Based on the known roles of the activated nuclei, it is suggested that, unlike the fish-diet, the soy-diet induced a potentially obesogenic profile, with high LH and low VMH/PVH activation after refeeding.

Patterns of fos activation in rat raphe nuclei during feeding behavior

To analyze the differential recruitment of the raphe nuclei during different phases of feeding behavior, rats were subjected to a food restriction schedule (food for 2 h/day, during 15 days). The animals were submitted to different feeding conditions, constituting the experimental groups: search for food (MFS), food ingestion (MFI), satiety (MFSa) and food restriction control (MFC). A baseline condition (BC) group was included as further control. The MFI and MFC groups, which presented greater autonomic and somatic activation, had more FOS-immunoreactive (FOS-IR) neurons. The MFI group presented more labeled cells in the linear (LRN) and dorsal (DRN) nuclei; the MFC group showed more labeling in the median (MRN), pontine (PRN), magnus (NRM) and obscurus (NRO) nuclei; and the MFSa group had more labeled cells in the pallidus (NRP). The BC exhibited the lowest number of reactive cells. The PRN presented the highest percentage of activation in the raphe while the DRN the lowest. Additional experiments revealed few double-labeled (FOS-IR+ 5-HT-IR) cells within the raphe nuclei in the MFI group, suggesting little serotonergic activation in the raphe during food ingestion. These findings suggest a differential recruitment of raphe nuclei during various phases of feeding behavior. Such findings may reflect changes in behavioral state (e.g., food-induced arousal versus sleep) that lead to greater motor activation, and consequently increased FOS expression. While these data are consistent with the idea that the raphe system acts as gain setter for autonomic and somatic activities, the functional complexity of the raphe is not completely understood. (c) 2008 Elsevier B.V. All rights reserved.

Angiotensin II induces superoxide generation via NAD(P)H oxidase activation in isolated rat pancreatic islets

Angiotensin II (Ang II) controls blood pressure, electrolyte balance, cell growth and vascular remodeling. Ang II activates NAD(P)H oxidase in several tissues with important function in the control of insulin secretion. Considering the concomitant occurrence of hypertension, insulin resistance and pancreatic B cell secretion impairment in the development of type II diabetes the aim of the present study was to evaluate the effect of ANG II on NAD(P)H oxidase activation in isolated pancreatic islets. We found that ANGII-induced superoxide generation via NAD(P)H oxidase activation and increased protein and mRNA levels of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)). (C) 2008 Elsevier B.V. All rights reserved.

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Activation of insulin and IGF-1 signaling pathways by melatonin through MT1 receptor in isolated rat pancreatic islets

Melatonin diminishes insulin release through the activation of MT1 receptors and a reduction in cAMP production in isolated pancreatic islets of neonate and adult rats and in INS-1 cells ( an insulin-secreting cell line). The pancreas of pinealectomized rats exhibits degenerative pathological changes with low islet density, indicating that melatonin plays a role to ensure the functioning of pancreatic beta cells. By using immunoprecipitation and immunoblotting analysis we demonstrated, in isolated rat pancreatic islets, that melatonin induces insulin growth factor receptor (IGF-R) and insulin receptor (IR) tyrosine phosphorylation and mediates the activities of the PI3K/AKT and MEK/ERKs pathways, which are involved in cell survival and growth, respectively. Thus, the effects of melatonin on pancreatic islets do not involve a reduction in cAMP levels only. This indoleamine may regulate growth and differentiation of pancreatic islets by activating IGF-I and insulin receptor signaling pathways.

Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation

Motor cortex stimulation (MCS) has been used to treat patients with neuropathic pain resistant to other therapeutic approaches; however, the mechanisms of pain control by MCS are still not clearly understood. We have demonstrated that MCS increases the nociceptive threshold of naive conscious rats, with opioid participation. In the present study, the effect of transdural MCS on neuropathic pain in rats subjected to chronic constriction injury of the sciatic nerve was investigated. In addition, the pattern of neuronal activation, evaluated by Fos and Zif268 immunolabel, was performed in the spinal cord and brain sites associated with the modulation of persistent pain. MCS reversed the mechanical hyperalgesia and allodynia induced by peripheral neuropathy. After stimulation, Fos immunoreactivity (Fos-IR) decreased in the dorsal horn of the spinal cord and in the ventral posterior lateral and medial nuclei of the thalamus, when compared to animals with neuropathic pain. Furthermore, the MCS increased the Fos-IR in the periaqueductal gray, the anterior cingulate cortex and the central and basolateral amygdaloid nuclei. Zif268 results were similar to those obtained for Fos, although no changes were observed for Zif268 in the anterior cingulate cortex and the central amygdaloid nucleus after MCS. The present findings suggest that MCS reverts neuropathic pain phenomena in rats, mimicking the effect observed in humans, through activation of the limbic and descending pain inhibitory systems. Further investigation of the mechanisms involved in this effect may contribute to the improvement of the clinical treatment of persistent pain. (c) 2010 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

A rise in arterial PCO(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the Botzinger/C1 region (Botz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 +/- 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Delta = +56 +8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the Botz/C1 reduced PND (Delta = +43 +/- 7%, +52 +/- 6% or +56 +/- 11%, respectively). MCPG also reduced sSND (Delta = +41 +/- 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the Botz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the Botz/C1.