Effects of 3 weeks GMP oral administration on glutamatergic parameters in mice neocortex

Overstimulation of the glutamatergic system (excitotoxicity) is involved in various acute and chronic brain diseases. Several studies support the hypothesis that guanosine-5'-monophosphate (GMP) can modulate glutamatergic neurotransmission. The aim of this study was to evaluate the effects of chronically administered GMP on brain cortical glutamatergic parameters in mice. Additionally, we investigated the neuroprotective potential of the GMP treatment submitting cortical brain slices to oxygen and glucose deprivation (OGD). Moreover, measurements of the cerebrospinal fluid (CSF) purine levels were performed after the treatment. Mice received an oral administration of saline or GMP during 3 weeks. GMP significantly decreases the cortical brain glutamate binding and uptake. Accordingly, GMP reduced the immunocontent of the glutamate receptors subunits, NR2A/B and GluR1 (NMDA and AMPA receptors, respectively) and glutamate transporters EAAC1 and GLT1. GMP treatment significantly reduced the immunocontent of PSD-95 while did not affect the content of Snap 25, GLAST and GFAP. Moreover, GMP treatment increased the resistance of neocortex to OGD insult. The chronic GMP administration increased the CSF levels of GMP and its metabolites. Altogether, these findings suggest a potential modulatory role of GMP on neocortex glutamatergic system by promoting functional and plastic changes associated to more resistance of mice neocortex against an in vitro excitotoxicity event.

Caffeic acid phenethyl ester reduces the activation of the nuclear factor kappa B pathway by high-fat diet-induced obesity in mice

Objective. The aim of this study was to investigate the effect of CAPE on the insulin signaling and inflammatory pathway in the liver of mice with high fat diet induced obesity. Material/Methods. Swiss mice were fed with standard chow or high-fat diet for 12-week. After the eighth week, animals in the HFD group with serum glucose levels higher than 200 mg/dL were divided into two groups, HFD and HFD receiving 30 mg/kg of CAPE for 4 weeks. After 12 weeks, the blood samples could be collected and liver tissue extracted for hormonal and biochemical measurements, and insulin signaling and inflammatory pathway analyzes. Results. The high-fat diet group exhibited more weight gain, glucose intolerance, and hepatic steatosis compared with standard diet group. The CAPE treatment showed improvement in glucose sensitivity characterized by an area under glucose curve similar to the control group in an oral glucose tolerance test Furthermore, CAPE treatment promoted amelioration in hepatic steatosis compared with the high-fat diet group. The increase in glucose sensitivity was associated with the improvement in insulin-stimulated phosphorylation of the insulin receptor substrate-2, followed by an increase in Akt phosphorylation. In addition, it was observed that CAPE reduced the induction of the inflammatory pathway, c-jun-N- terminal kinase, the nuclear factor kappa B, and cyclooxygenase-2 expression, respectively. Conclusions. Overall, these findings indicate that CAPE exhibited anti-inflammatory activity that partly restores normal metabolism, reduces the molecular changes observed in obesity and insulin resistance, and therefore has a potential as a therapeutic agent in obesity. (C) 2012 Elsevier Inc. All rights reserved.

The role of cytoskeleton, components of inositol phospholipid signaling pathway and iron in Ehrlichia canis in vitro proliferation

Ehrlichia canis, etiologic agent of Canine Monocytic Ehrlichiosis, is an obligatory intracellular bacterium that parasitizes monocytes and macrophages. In this study we analyzed the role of the cytoskeleton specifically actin microfilaments and microtubules, components of inositol phospholipid signaling pathway such as phospholipase C (PLC), protein kinase (PTK) and calcium channels as well as the role of iron in the E. canis proliferation in DH82 cells. Different inhibitory compounds were used for each component: Cytochalasin D (inhibits actin polymerization), Nocodazole (inhibits microtubule polymerization), Neomycin (PLC inhibitor), Genistein (PTK inhibitor), Verapamil (calcium channel blocker) and Deferoxamine (iron chelator). We observed a significant decrease in the total number of bacteria in infected cells treated suggesting that these cellular components analized are essentials to E. canis proliferation.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: An EEG wavelet analysis and retrograde neuronal tracer approach

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG + projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background. (C) 2012 Elsevier Inc. All rights reserved.

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/reperfusion conditions following cilostazol administration. METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence. RESULTS: Acetylcholine- and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/Cilostazol/Reperfusion groups. CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.

Post-receptor IGF1 insensitivity restricted to the MAPK pathway in a Silver-Russell syndrome patient with hypomethylation at the imprinting control region on chromosome 11

Background: Hypomethylation of the paternal imprinting center region 1 (ICR1) is the most frequent molecular cause of Silver-Russell syndrome (SRS). Clinical evidence suggests that patients with this epimutation have mild IGF1 insensitivity. Objective: To assess in vitro IGF1 action in fibroblast culture from a patient with SRS and IGF1 insensitivity. Methods: Fibroblast cultures from one patient with SRS due to ICR1 demethylation and controls were established. The SRS patient has severe growth failure, elevated IGF1 level, and poor growth rate during human recombinant GH treatment. IGF1 action was assessed by cell proliferation, AKT, and p42/44-MAPK phosphorylation. Gene expression was determined by real-time PCR. Results: Despite normal IGF1R sequence and expression, fibroblast proliferation induced by IGF1 was 50% lower in SRS fibroblasts in comparison with controls. IGF1 and insulin promoted a p42/44-MAPK activation in SRS fibroblasts 40 and 36%, respectively, lower than that in control fibroblasts. On the other hand, p42/44-MAPK activation induced by EGF stimulation was only slightly reduced (75% in SRS fibroblasts in comparison with control), suggesting a general impairment in MAPK pathway with a greater impairment of the stimulation induced by insulin and IGF1 than by EGF. A PCR array analysis disclosed a defect in MAPK pathway characterized by an increase in DUSP4 and MEF2C gene expressions in patient fibroblasts. Conclusion: A post-receptor IGF1 insensitivity was characterized in one patient with SRS and ICR1 hypomethylation. Although based on one unique severely affected patient, these results raise an intriguing mechanism to explain the postnatal growth impairment observed in SRS patients that needs confirmation in larger cohorts.

Exogenous ornithine is an effective precursor and the delta-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the delta-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Delta(1)-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS. GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Delta(1)-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Oxidation of lysergic acid diethylamide (LSD) by peroxidases: a new metabolic pathway

Lysergic acid diethylamide (LSD) is a potent hallucinogen that is primarily metabolized to 2-oxo-3-hydroxy-LSD (O-H-LSD) and N-desmethyl-LSD (nor-LSD) by cytochrome P450 complex liver enzymes. Due to its extensive metabolism, there still is an interest in the identification of new metabolites and new routes of its metabolism in humans. In the present study, we investigated whether LSD could be a substrate for horseradish peroxidase or myeloperoxidase (MPO). Using liquid chromatography coupled to UV detection and electrospray ionization mass spectrometry (LC-UV-ESI-MS), we found that both peroxidases were capable of metabolizing LSD to the same compounds that have been observed in vivo (i.e., O-H-LSD and nor-LSD). In addition, we found another major metabolite, N,N-diethyl-7-formamido-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide (FOMBK), which is an opened indolic ring compound. Hydrolysis of FOMBK led to the deformylated compound 7-amino-N,N-diethyl-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide. The reactions of LSD with the peroxidases were chemiluminescent and sensitive to inhibition by reactive oxygen scavengers, which indicated that the classic peroxidase cycle is involved in this new alternative metabolic pathway. Considering that MPO is abundant in immune cells and also present in the central nervous system, the degradation pathway described in this study suggests a possible route of LSD metabolism that may occur concurrently with the in vivo reaction catalyzed by the cytochrome P450 system.

TLR2, TLR4 and the MYD88 Signaling Pathway Are Crucial for Neutrophil Migration in Acute Kidney Injury Induced by Sepsis

The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-), TLR4(-/-) and MyD88(-/-) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2(-/-), TLR4(-/-) and MyD88(-/-) mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88(-/-) mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1(+low) cells migration compared with the knockout mice and decreased in GR1(+high) cells migration into the peritoneal cavity. The TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway

Background Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. Objective Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. Methods We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. Results We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. Conclusions and Clinical Relevance Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen-and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-kappa B pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-kappa B) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-kappa B DNA binding activity (NF-kappa Bp50 and NF-kappa Bp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-kappa B pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-kappa B (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-kappa B (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-kappa B signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals. (C) 2011 Elsevier Ltd. All rights reserved.

Proteomic Approaches Identify Members of Cofilin Pathway Involved in Oral Tumorigenesis

The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one-and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the "more-aggressive'' group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the "less-aggressive'' group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas.

Trypanosoma cruzi invades host cells through the activation of endothelin and bradykinin receptors: a converging pathway leading to chagasic vasculopathy

BACKGROUND AND PURPOSE Independent studies in experimental models of Trypanosoma cruzi appointed different roles for endothelin-1 (ET-1) and bradykinin (BK) in the immunopathogenesis of Chagas disease. Here, we addressed the hypothesis that pathogenic outcome is influenced by functional interplay between endothelin receptors (ETAR and ETBR) and bradykinin B2 receptors (B2R). EXPERIMENTAL APPROACH Intravital microscopy was used to determine whether ETR/B2R drives the accumulation of rhodamine-labelled leucocytes in the hamster cheek pouch (HCP). Inflammatory oedema was measured in the infected BALB/c paw of mice. Parasite invasion was assessed in CHO over-expressing ETRs, mouse cardiomyocytes, endothelium (human umbilical vein endothelial cells) or smooth muscle cells (HSMCs), in the presence/absence of antagonists of B2R (HOE-140), ETAR (BQ-123) and ETBR (BQ-788), specific IgG antibodies to each GPCRs; cholesterol or calcium-depleting drugs. RNA interference (ETAR or ETBR genes) in parasite infectivity was investigated in HSMCs. KEY RESULTS BQ-123, BQ-788 and HOE-140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ETAR and ETBR antagonists reduced parasite invasion of HSMCs to the same extent as HOE-140. Exogenous ET-1 potentiated T. cruzi uptake by HSMCs via ETRs/B2R, whereas RNA interference of ETAR and ETBR genes conversely reduced parasite internalization. ETRs/B2R-driven infection in HSMCs was reduced in HSMC pretreated with methyl-beta-cyclodextrin, a cholesterol-depleting drug, or in thapsigargin-or verapamil-treated target cells. CONCLUSIONS AND IMPLICATIONS Our findings suggest that plasma leakage, a neutrophil-driven inflammatory response evoked by trypomastigotes via the kinin/endothelin pathways, may offer a window of opportunity for enhanced parasite invasion of cardiovascular cells.

Inhibition of the NMDA receptor/Nitric Oxide pathway in the dorsolateral periaqueductal gray causes anxiolytic-like effects in rats submitted to the Vogel conflict test

Abstract Background Several studies had demonstrated the involvement of the dorsolateral portion of periaqueductal grey matter (dlPAG) in defensive responses. This region contains a significant number of neurons containing the enzyme nitric oxide synthase (NOS) and previous studies showed that non-selective NOS inhibition or glutamate NMDA-receptor antagonism in the dlPAG caused anxiolytic-like effects in the elevated plus maze. Methods In the present study we verified if the NMDA/NO pathway in the dlPAG would also involve in the behavioral suppression observed in rats submitted to the Vogel conflict test. In addition, the involvement of this pathway was investigated by using a selective nNOS inhibitor, Nω-propyl-L-arginine (N-Propyl, 0.08 nmol/200 nL), a NO scavenger, carboxy-PTIO (c-PTIO, 2 nmol/200 nL) and a specific NMDA receptor antagonist, LY235959 (4 nmol/200 nL). Results Intra-dlPAG microinjection of these drugs increased the number of punished licks without changing the number of unpunished licks or nociceptive threshold, as measure by the tail flick test. Conclusion The results indicate that activation of NMDA receptors and increased production of NO in the dlPAG are involved in the anxiety behavior displayed by rats in the VCT.

Preimplantation development regulatory pathway construction through a text-mining approach

Background: The integration of sequencing and gene interaction data and subsequent generation of pathways and networks contained in databases such as KEGG Pathway is essential for the comprehension of complex biological processes. We noticed the absence of a chart or pathway describing the well-studied preimplantation development stages; furthermore, not all genes involved in the process have entries in KEGG Orthology, important information for knowledge application with relation to other organisms. Results: In this work we sought to develop the regulatory pathway for the preimplantation development stage using text-mining tools such as Medline Ranker and PESCADOR to reveal biointeractions among the genes involved in this process. The genes present in the resulting pathway were also used as seeds for software developed by our group called SeedServer to create clusters of homologous genes. These homologues allowed the determination of the last common ancestor for each gene and revealed that the preimplantation development pathway consists of a conserved ancient core of genes with the addition of modern elements. Conclusions: The generation of regulatory pathways through text-mining tools allows the integration of data generated by several studies for a more complete visualization of complex biological processes. Using the genes in this pathway as “seeds” for the generation of clusters of homologues, the pathway can be visualized for other organisms. The clustering of homologous genes together with determination of the ancestry leads to a better understanding of the evolution of such process.