Analytical method for urinary homovanillic acid and 5-hydroxyindoleacetic acid levels using HPLC with electrochemical detection applied to evaluate children environmentally exposed to manganese.

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) are the urinary metabolites of dopamine (DA) and serotonin (5-HA), respectively. We aimed to develop an extraction method for the determination of HVA and 5-HIAA, using strong anionic exchange cartridges combined with HPLC with electrochemical detection, and apply it to measure the levels of HVA and 5-HIAA in children living near a ferro-manganese alloy plant in Simões Filho, Brazil. The validated method showed good selectivity, sensitivity, precision, and accuracy. The limits of detection (LOD) were 4 and 8 µmol/L for 5-HIAA and HVA, respectively, in urine. Recoveries ranged from 85.8 to 94%. The coefficients of determination (R2 ) of the calibration curves were greater than 0.99. Spot urine samples of 30 exposed children and 20 nonexposed ones were processed accordingly. The metabolite levels in exposed and reference children were within the physiological ranges. The medians (range) for 5-HIAA and HVA of the exposed ones were 36.4 µmol/L (18.4-58.0) and 32.9 µmol/L (

A Sea Anemone Lebrunia neglecta Venom Fraction Decreases Boar Sperm Cells Capacitation: Possible Involvement of HVA Calcium Channels.

Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells' current kinetics and current-voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders.

Reactive species play an important role in physiological functions. Overproduction of reactive species, notably reactive oxygen (ROS) and nitrogen (RNS) species along with the failure of balance by the body's antioxidant enzyme systems results in destruction of cellular structures, lipids, proteins, and genetic materials such as DNA and RNA. Moreover, the effects of reactive species on mitochondria and their metabolic processes eventually cause a rise in ROS/RNS levels, leading to oxidation of mitochondrial proteins, lipids, and DNA. Oxidative stress has been considered to be linked to the etiology of many diseases, including neurodegenerative diseases (NDDs) such as Alzheimer diseases, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Multiple sclerosis, and Parkinson's diseases. In addition, oxidative stress causing protein misfold may turn to other NDDs include Creutzfeldt-Jakob disease, Bovine Spongiform Encephalopathy, Kuru, Gerstmann-Straussler-Scheinker syndrome, and Fatal Familial Insomnia. An overview of the oxidative stress and mitochondrial dysfunction-linked NDDs has been summarized in this review.

Rimonabant improves metabolic parameters partially attributed to restoration of high voltage-activated Ca2+ channels in skeletal muscle in HFD-fed mice

Cannabinoid type 1 receptor (CB1R) inhibition tends to be one of the promising strategies for the treatment of obesity and other related metabolic disorders. Although CB1R inhibition may cause adverse psychiatric effects including depression and anxiety, the investigation of the role of peripheral CB1R on weight loss and related metabolic parameters are urgently needed. We first explored the effect of rimonabant, a selective CB1R antagonist/inverse agonist, on some metabolic parameters in high fat-diet (HFD)-induced obesity in mice. Then, real-time PCR and electrophysiology were used to explore the contribution of high voltage-activated Ca2+ channels (HVACCs), especially Cav1.1, on rimonabant's effect in skeletal muscle (SM) in HFD-induced obesity. Five-week HFD feeding caused body weight gain, and decreased glucose/insulin tolerance in mice compared to those in the regular diet group (P<0.05), which was restored by rimonabant treatment compared to the HFD group (P<0.05). Interestingly, HVACCs and Cav1.1 were decreased in soleus muscle cells in the HFD group compared to the control group. Daily treatment with rimonabant for 5 weeks was shown to counter such decrease (P<0.05). Collectively, our findings provided a novel understanding for peripheral CB1R's role in the modulation of body weight and glucose homeostasis and highlight peripheral CB1R as well as Cav1.1 in the SM as potential targets for obesity treatment.

The MAP1B-LC1/UBE2L3 complex catalyzes degradation of cell surface CaV2.2 channels.

We reported recently a new mechanism by which the neuronal N-type Ca(2+) (CaV2.2) channel expression may be regulated by ubiquitination. This mechanism involves the interaction between the channel and the light chain (LC1) of the microtubule associated protein B (MAP1B). We also showed that MAP1B-LC1 could interact with the ubiquitin-conjugating E2 enzyme UBE2L3 and that the ubiquitination/degradation mechanism triggered by MAP1B-LC1 could be prevented by inhibiting the ubiquitin-proteasome proteolytic pathway. We now report that MAP1B-LC1 can interact with the 2 main variants of the CaV2.2 channels (CaV2.2e37a and CaV2.2e37b) and that the MAP1B-LC1-mediated regulation most likely involves an internalization of the channels via a dynamin and clathrin-dependent pathway. In addition, here we propose that this novel mechanism of CaV channel regulation might be conserved among N-type and P/Q-type channels.

Dopaminergic D2 receptor is a key player in the substantia nigra pars compacta neuronal activation mediated by REM sleep deprivation.

Currently, several studies addresses the novel link between sleep and dopaminergic neurotransmission, focusing most closely on the mechanisms by which Parkinson's disease (PD) and sleep may be intertwined. Therefore, variations in the activity of afferents during the sleep cycles, either at the level of DA cell bodies in the ventral tegmental area (VTA) and/or substantia nigra pars compacta (SNpc) or at the level of dopamine (DA) terminals in limbic areas may impact functions such as memory. Accordingly, we performed striatal and hippocampal neurochemical quantifications of DA, serotonin (5-HT) and metabolites of rats intraperitoneally treated with haloperidol (1.5 mg/kg) or piribedil (8 mg/kg) and submitted to REM sleep deprivation (REMSD) and sleep rebound (REB). Also, we evaluated the effects of REMSD on motor and cognitive parameters and SNpc c-Fos neuronal immunoreactivity. The results indicated that DA release was strongly enhanced by piribedil in the REMSD group. In opposite, haloperidol prevented that alteration. A c-Fos activation characteristic of REMSD was affected in a synergic manner by piribedil, indicating a strong positive correlation between striatal DA levels and nigral c-Fos activation. Hence, we suggest that memory process is severely impacted by both D2 blockade and REMSD and was even more by its combination. Conversely, the activation of D2 receptor counteracted such memory impairment. Therefore, the present evidence reinforce that the D2 receptor is a key player in the SNpc neuronal activation mediated by REMSD, as a consequence these changes may have direct impact for cognitive and sleep abnormalities found in patients with PD. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'.

Neuroprotective effects of swimming training in a mouse model of Parkinson's disease induced by 6-hydroxydopamine.

Parkinson's disease (PD) is characterized by progressive dopamine (DA) depletion in the striatum. Exercise has been shown to be a promising non-pharmacological approach to reduce the risk of neurodegeneration diseases. This study was designed to investigate the potential neuroprotective effect of swimming training (ST) in a mouse model of PD induced by 6-hydroxydopamine (6-OHDA) in mice. The present study demonstrated that a 4-week ST was effective in attenuating the following impairments resulting from 6-OHDA exposure: (i) depressive-like behavior in the tail suspension test; (ii) increase in the number of falls in the rotarod test; (iii) impairment on long-term memory in the object recognition test; (iv) increase of the reactive species and interleukin 1-beta (IL-1ß) levels; (v) inhibition of the glutathione peroxidase (GPx) activity; (vi) rise of the glutathione reductase (GR) and glutathione S-transferase (GST) activities and vii) decrease of DA, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels. The mechanisms involved in this study are the modulation of GPx, GR and GST activities as well as IL-1ß level in a PD model induced by 6-OHDA, protecting against the decrease of DA, DOPAC and HVA levels in the striatum of mice. These findings reinforce that one of the effects induced by exercise on neurodegenerative disease, such as PD, is due to antioxidant and anti-inflammatory properties. We suggest that exercise attenuates cognitive and motor declines, depression, oxidative stress, and neuroinflammation induced by 6-OHDA supporting the hypothesis that exercise can be used as a non-pharmacological tool to reduce the symptoms of PD.

Sex-dependent effects of maternal separation on plasma corticosterone and brain monoamines in response to chronic ethanol administration.

Prolonged and repeated periods of maternal separation produce behavioral phenotype of increased vulnerability to neuropsychiatric disorders and drug abuse. Most of the changes in behavior, corticosterone (CORT) and monoamine levels induced by long maternal separation (LMS) are observed after a challenge, but not in basal conditions. LMS increases ethanol-induced locomotor response and self-administration, possibly due to changes in CORT release and/or monoamine concentrations. This study examined the effects of LMS in association with chronic ethanol treatment on plasma CORT and brain monoamine concentrations in male and female Swiss mice, which were kept undisturbed (animal facility rearing - AFR) or separated from their mothers for 3h/day, from 2 to 14 days of age (LMS). As adults, one set of male and female mice received no drug treatment to assess the effect of LMS per se. Another set of animals received saline injections for 20 days and one ethanol injection (2.2g/kg, i.p.) on day 21 (acute) or ethanol for 21 days (chronic). Locomotor activity, plasma CORT levels and monoamines in the frontal cortex, striatum and hippocampus of AFR and LMS mice were evaluated in non-treated, acute and chronic ethanol-treated animals. In non-treated mice, no differences were found in CORT or locomotor activity, with small changes in monoamines content. In LMS females, chronic ethanol increased dopamine and serotonin concentrations in the frontal cortex, relative to acute ethanol LMS and to chronic ethanol-treated AFR groups (p<0.05). In LMS males, chronic ethanol increased hippocampal noradrenaline, dopamine, serotonin and metabolites when compared to respective AFR controls, as well as acute LMS. Moreover, chronic ethanol treatment resulted in higher CORT concentrations in LMS than in AFR males. Overall, these results indicate that LMS mice were more susceptible to the effects of chronic ethanol administration on CORT and brain monoamine concentrations, and that these effects were sex-dependent.

Ascending monoaminergic systems alterations in Alzheimer's disease. translating basic science into clinical care.

Extensive neuropathological studies have established a compelling link between abnormalities in structure and function of subcortical monoaminergic (MA-ergic) systems and the pathophysiology of Alzheimer's disease (AD). The main cell populations of these systems including the locus coeruleus, the raphe nuclei, and the tuberomamillary nucleus undergo significant degeneration in AD, thereby depriving the hippocampal and cortical neurons from their critical modulatory influence. These studies have been complemented by genome wide association studies linking polymorphisms in key genes involved in the MA-ergic systems and particular behavioral abnormalities in AD. Importantly, several recent studies have shown that improvement of the MA-ergic systems can both restore cognitive function and reduce AD-related pathology in animal models of neurodegeneration. This review aims to explore the link between abnormalities in the MA-ergic systems and AD symptomatology as well as the therapeutic strategies targeting these systems. Furthermore, we will examine possible mechanisms behind basic vulnerability of MA-ergic neurons in AD.

Modulation of post-coital dopaminergic tone by prior parity

The aim of this study was to investigate the possible effects of reproductive experience on dopaminergic profile in three different brain tissues, hypothalamus, striatum and cortex in rats on 7th-8th day of pregnancy during the light-dark shift (between 1700-1900h). Results showed that in hypothalamus, dopamine levels increased and DOPAC/DA decreased as a function of parity. In cortex, no differences were observed. In striata, the haloperidol-induced HVA and HVA/DA increases were less intense in experienced animals. These findings suggested that reproductive experience produced functional central changes during pregnancy, with different neurochemical responses depending on the brain region.

A dopamina age nos níveis endócrino, neuroquímico e comportamental. A experiência reprodutiva modula alguns destes aspectos: a dopamina está aumentada no estriato e no hipotálamo de ratas muiltigrávidas entre 12:00-14:00 horas. A sensibilidade dos terminais dopaminérgicos também parece ser modulada por uma experiência reprodutiva prévia. Nosso objetivo foi o de investigar os possíveis efeitos da experiência reprodutiva no perfil dopaminérgico de três diferentes tecidos cerebrais, hipotálamo, estriato e córtex em ratas no 7º-8º dia de gestação durante a fase de virada do ciclo claro-escuro (entre 17:00-19:00h). Nossos resultados mostraram que os níveis dopaminérgicos no hipotálamo aumentaram e o índice DOPAC/DA diminuiu em multigrávidas quando comparadas às primigrávidas. No córtex, nenhuma diferença foi encontrada. No estriato, o aumento de HVA e HVA/DA induzidos pelo haloperidol foi menos intenso nos animais experientes. Nossos resultados sugerem que a experiência reprodutiva produz alterações funcionais centrais durante a gestação, com diferentes respostas neuroquímicas de acordo com a região cerebral.

Determinação simultânea de VMA, 5HIAA e HVA urinários sem extração / Simultaneous determination of urinary VMA, 5HIAA and HVA without extraction

Este trabalho mostra as vantagens de se determinar simultaneamente os metabólitos VMA, 5HIAA, HVA urinários. A quantificação destes metabólitos são muito importantes nos diagnósticos e tratamento de tumores que se originam na crista neural (VMA, HVA), e diagnóstico da síndrome e tumor carcinóide (5HIAA). A maioria das técnicas utilizadas, limitam-se a medir somente um ou dois setes metabólitos por análise, sendo que a ESA atualmente desenvolveu um procedimento direto, onde se obtém uma quantificação simultânea dos três metabólitos (VMA, 5HIAA, HVA) urinários usando o Sistema Coulochem Electrode Array – CEAS