Effects of neonatal handling on central noradrenergic and nitric oxidergic systems and reproductive parameters in female rats

Early-life environmental events that disrupt the mother-pup relationship may induce profound long-lasting changes on several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces sexual behavior and induces anovulatory estrous cycles in female rats. On the afternoon of proestrus, neonatally handled females show a reduced surge of luteinizing hormone (LH) and an increased content of gonadotropin-releasing hormone in the medial preoptic area (MPOA). In order to detect the possible causes for the reduced ovulation and sexual behavior, the present study aimed to analyze the effects of neonatal handling on noradrenaline (NA) and nitric oxide (NO) levels in the MPOA on the afternoon of proestrus. Neonatal handling reduced MHPG (NA metabolite) levels and MHPG/NA ratio in the MPOA, indicating decreased NAergic activity. Additionally, neonatal handling decreased NO levels, as measured by the metabolites (NO x), nitrite and nitrate in the same period. We may conclude that the neonatal handling procedure decreased activity of the NAergic and NOergic systems in the MPOA during proestrus, which is involved in the control of LH and FSH secretion, and this may possibly explain the anovulatory estrous cycles and reduced sexual behavior of the neonatally handled female rats. Copyright (c) 2007 S. Karger AG, Basel.

Serotonergic mechanisms on breathing modulation in the rat locus coeruleus

The locus coeruleus (LC) is a noradrenergic nucleus that plays an important role in the ventilatory response to hypercapnia. This nucleus is densely innervated by serotonergic fibers and contains high density of serotonin (5-HT) receptors, including 5-HT(1A) and 5-HT(2). We assessed the possible modulation of respiratory response to hypercapnia by 5-HT, through 5-HT(1A) and 5-HT(2) receptors, in the LC. To this end, we determined the concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the LC after hypercapnic exposure. Pulmonary ventilation (V(E), plethysmograph) was measured before and after unilateral microinjection (100 nL) of WAY-100635 (5-HT(1A) antagonist, 5.6 and 56 mM), 8-OHDPAT (5-HT(1A/7) agonist, 7 and 15 mM), Ketanserin (5-HT(2A) antagonist, 3.7 and 37 mM), or (+/-)-2,5-dimethoxy-4-iodoamphetaminehydrochloride (DOI; 5-HT(2A) agonist, 6.7 and 67 mM) into the LC, followed by a 60-min period of 7% CO(2) exposure. Hypercapnia increased 5-HTIAA levels and 5-HIAA/5-HT ratio within the LC. WAY-100635 and 8-OHDPAT intra-LC decreased the hypercapnic ventilatory response due to a lower tidal volume. Ketanserin increased CO(2) drive to breathing and DOI caused the opposite response, both acting on tidal volume. The current results provide evidence of increased 5-HT release during hypercapnia in the LC and that 5-HT presents an inhibitory modulation of the stimulatory role of LC on hypercapnic ventilatory response, acting through postsynaptic 5-HT(2A) receptors in this nucleus. In addition, hypercapnic responses seem to be also regulated by presynaptic 5-HT(1A) receptors in the LC.

Noradrenaline Involvement in the Negative-Feedback Effects of Ovarian Steroids on Luteinising Hormone Secretion

Noradrenaline has been shown to modulate the ovarian-steroid feedback on luteinising-hormone (LH) release. However, despite the high amount of evidence accumulated over many years, the role of noradrenaline in LH regulation is still not clearly understood. The present study aimed to further investigate the involvement of noradrenaline in the negative-feedback effect of oestradiol and progesterone on basal LH secretion. In experiment 1, ovariectomised (OVX) rats received a single injection of oil, oestradiol, or progesterone at 09.00-10.00 h and were decapitated 30 or 60 min later. Levels of noradrenaline and its metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), were determined in microdissections of the preoptic area (POA) and medial basal hypothalamus-median eminence (MBH-ME) and correlated with LH secretion. Basal LH levels were decreased 30 and 60 min after oestradiol or progesterone injection, and this hormonal response was significantly correlated with a reduction in POA MHPG levels, which reflect noradrenaline release. In addition, noradrenaline levels in the POA were increased, whereas noradrenaline turnover (MHPG/noradrenaline ratio) was decreased 60 min after the injection of both hormones. No effect was found in the MBH-ME. In experiment 2, i.c.v. administration of noradrenaline (60 nmol), performed 15 min before oestradiol or progesterone injection in jugular vein-cannulated OVX rats, completely prevented the ovarian steroid-induced inhibition of LH secretion. The data obtained provide direct evidence that LH secretion in OVX rats is positively regulated by basal noradrenergic activity in the POA, and its reduction appears to play a role in the negative-feedback effect of ovarian steroids on LH secretion in vivo.

Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO(2) concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana: a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate. (C) 2010 Elsevier GmbH. All rights reserved.

Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K-m=0.59 +/- 0.04 mM, V-max=0.123 +/- 0.003 nmol/min per 20 x 10(6) cells) showing an energy of activation of 52.38 +/- 4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, alpha-ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H+ but not to Na+ or K+. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K+, and not H+ membrane gradient in the transport process.

Annual profile of fecal androgen and glucocorticoid levels in free-living male American kestrels from southern mid-latitude areas

Fecal samples and behavioral data were collected at a fortnightly basis during 11 months period from free-living male American kestrels living in southeast Brazil (22 degrees S latitude). The aim was to investigate the seasonal changes in testicular and adrenal steroidogenic activity and their correlation to reproductive behaviors and environmental factors. The results revealed that monthly mean of fecal glucocorticoid metabolites in May and June were higher than those estimated in November. in parallel, monthly mean of androgen metabolites in September was higher than those from January to April and from October to November. Molt took place from January to March, whereas copulation was observed from June to October but peaked in September. Nest activity and food transfer to females occurred predominantly in October, and parental behavior was noticed only in November. Territorial aggressions were rare and scattered throughout the year. Multiple regression analysis revealed that fecal androgen levels are predicted by photoperiod and copulation, while fecal glucocorticoid levels are only predicted by photoperiod. Bivariate correlations showed that fecal androgen metabolites were positively correlated with fecal glucocorticoid metabolites and copulation, but negatively correlated with molt. Additionally, copulation was positively correlated with food transfer to females and nest activity, but negatively correlated with molt. These findings suggest that male American kestrels living in southeast Brazil exhibit significant seasonal changes in fecal androgen and glucocorticoid concentrations, which seem to be stimulated by decreasing daylength but not by rainfall or temperature. (C) 2009 Elsevier Inc. All rights reserved.

Chronic supplementation of beta-hydroxy-beta methylbutyrate (HM beta) increases the activity of the GH/IGF-I axis and induces hyperinsulinemia in rats

Objective: Beta-hydroxy-beta-methylbutyrate (HM beta) is a metabolite of leucine widely used for improving sports performance. Although limp is recognized to promote anabolic or anti-catabolic effects on protein metabolism, the impact of its long-term use on skeletal muscle and/or genes that control the skeletal protein balance is not fully known. This study aimed to investigate whether chronic HM beta treatment affects the activity of GH/IGF-I axis and skeletal muscle IGF-I and myostatin mRNA expression. Design: Rats were treated with HK beta (320 mg/kg BW) or vehicle, by gavage, for 4 weeks, and killed by decapitation. Blood was collected for evaluation of serum insulin, glucose and IGF-I concentrations. Samples of pituitary, liver, extensor digitorum longus (EDL) and soleus muscles were collected for total RNA or protein extraction to evaluate the expression of pituitary growth hormone (GH) gene (mRNA and protein), hepatic insulin-like growth factor I (IGF-I) mRNA, skeletal muscle IGF-I and myostatin mRNA by Northern blotting/real time-PCR, or Western blotting. Results: Chronic HM beta treatment increased the content of pituitary GH mRNA and GH, hepatic IGF-I mRNA and serum IGF-I concentration. No changes were detected on skeletal muscle IGF-I and myostatin mRNA expression. However, the HIM-treated rats although normoglycemic, exhibited hyperinsulinemia. Conclusions: The data presented herein extend the body of evidence on the potential role of HM beta-treatment in stimulating GH/IGF-I axis activity. In spite of this effect, HM beta supplementation also induces an apparent insulin resistance state which might limit the beneficial aspects of the former results, at least in rats under normal nutritional status and health conditions. (C) 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

Quantification of chlordesmethyldiazepam by liquid chromatography-tandem mass spectrometry: application to a cloxazolam bioequivalence study

A rapid, sensitive and specific LC-MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid-liquid extraction (diethyl-ether/hexane, 80/20, v/v) procedure. The LC-MS/MS method on a RP-C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5-50 ng/mL (R > 0.999). The between-run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification-LLOQ (0.500 ng/mL). The between-run accuracies were 0.1, -1.5, -2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam-test, Eurofarma Lab. Ltda and Olcadil (R)-reference, Novartis Biociencias S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUCO-inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA. Copyright (C) 2009 John Wiley & Sons, Ltd.

Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice

Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor L-NAME in ovalbumin (OVA)-challenged mice. Acute L-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF In contrast, 3-week L-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week L-NAME treatments. The pulmonary iNOS activity was reduced by acute L-NAME (or aminoguanidine), but unaffected by 3-week L-NAME treatment. Acute L-NAME (or aminoguanidine) treatment was more efficient to reduce the NO(x) levels compared with 3-week L-NAME treatment. The pharmacokinetic study revealed that L-NAME is not bioavailable when given orally. After acute L-NAME intake, serum concentrations of the metabolite N(omega)-nitro-L-arginine decreased from 30 min to 24 h. In the 3-week L-NAME treatment, the N(omega)-nitro-L-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with L-NAME yields low serum N(omega)-nitro-L-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week L-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO. (c) 2008 Elsevier Ltd. All rights reserved.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1 Delta tsa2 Delta) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1 Delta tsa2 Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1 Delta tsa2 Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu, Zn-superoxide dismutase (Sod1), whose expression and activity increased similar to 5- and 2-fold, respectively, in tsa1 Delta tsa2 Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1 Delta tsa2 Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1 Delta tsa2 Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

cis-4-decenoic acid provokes mitochondrial bioenergetic dysfunction in rat brain

Aims: In the present work we investigated the in vitro effect of cis-4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods: Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings: cis-4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis-4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis-4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondria! membrane. Significance: It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis-4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients. (C) 2010 Elsevier Inc. All rights reserved.

Resolution and Absolute Configuration Assignment of a Natural Racemic Chromane from Peperomia obtusifolia (Piperaceae)

The resolution of the natural racemic chromane 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(3 ``-methyl-2 ``-butenyl)-2-(4`-methyl-1`,3`-pentadienyl)-2H-1-benzopyran-6-carboxylic acid (1) isolated from the leaves of Peperomia obtusifolia has been accomplished using stereoselective HPLC. The absolute coil figuration of the resolved enantiomers was determined by the analysis of optical rotations and CD spectra. The finding of a racemic mixture instead of an enantiomerically pure metabolite raises questions about the final steps in the biosynthesis of this class of natural products, suggesting that the intramolecular chromane ring formation step may not be enzymatically controlled at all in P. obtusifolia. Chirality 21:799-801, 2009. (C) 2008 Wiley-Liss, Inc.

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l(-1)) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l(-1) 2,4-D and 2 mg.l(-1) BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.