Fine-tuning of defensive behaviors in the dorsal periaqueductal gray by atypical neurotransmitters

This paper presents an up-to-date review of the evidence indicating that atypical neurotransmitters such as nitric oxide (NO) and endocannabinoids (eCBs) play an important role in the regulation of aversive responses in the periaqueductal gray (PAG). Among the results supporting this role, several studies have shown that inhibitors of neuronal NO synthase or cannabinoid receptor type 1 (CB1) receptor agonists cause clear anxiolytic responses when injected into this region. The nitrergic and eCB systems can regulate the activity of classical neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) that control PAG activity. We propose that they exert a ‘fine-tuning’ regulatory control of defensive responses in this area. This control, however, is probably complex, which may explain the usually bell-shaped dose-response curves observed with drugs that act on NO- or CB1-mediated neurotransmission. Even if the mechanisms responsible for this complex interaction are still poorly understood, they are beginning to be recognized. For example, activation of transient receptor potential vanilloid type-1 channel (TRPV1) receptors by anandamide seems to counteract the anxiolytic effects induced by CB1 receptor activation caused by this compound. Further studies, however, are needed to identify other mechanisms responsible for this fine-tuning effect.

Mapping and signaling of neural pathways involved in the regulation of hydromineral homeostasis

Several forebrain and brainstem neurochemical circuitries interact with peripheral neural and humoral signals to collaboratively maintain both the volume and osmolality of extracellular fluids. Although much progress has been made over the past decades in the understanding of complex mechanisms underlying neuroendocrine control of hydromineral homeostasis, several issues still remain to be clarified. The use of techniques such as molecular biology, neuronal tracing, electrophysiology, immunohistochemistry, and microinfusions has significantly improved our ability to identify neuronal phenotypes and their signals, including those related to neuron-glia interactions. Accordingly, neurons have been shown to produce and release a large number of chemical mediators (neurotransmitters, neurohormones and neuromodulators) into the interstitial space, which include not only classic neurotransmitters, such as acetylcholine, amines (noradrenaline, serotonin) and amino acids (glutamate, GABA), but also gaseous (nitric oxide, carbon monoxide and hydrogen sulfide) and lipid-derived (endocannabinoids) mediators. This efferent response, initiated within the neuronal environment, recruits several peripheral effectors, such as hormones (glucocorticoids, angiotensin II, estrogen), which in turn modulate central nervous system responsiveness to systemic challenges. Therefore, in this review, we shall evaluate in an integrated manner the physiological control of body fluid homeostasis from the molecular aspects to the systemic and integrated responses.

Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition

The present study investigated the effect of silibinin, the principal potential anti-inflammatory flavonoid contained in silymarin, a mixture of flavonolignans extracted from Silybum marianum seeds, on palmitate-induced insulin resistance in C2C12 myotubes and its potential molecular mechanisms. Silibinin prevented the decrease of insulin-stimulated 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) uptake and the downregulation of glutamate transporter type 4 (GLUT4) translocation in C2C12 myotubes induced by palmitate. Meanwhile, silibinin suppressed the palmitate-induced decrease of insulin-stimulated Akt Ser473 phosphorylation, which was reversed by wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K). We also found that palmitate downregulated insulin-stimulated Tyr632 phosphorylation of insulin receptor substrate 1 (IRS-1) and up-regulated IRS-1 Ser307 phosphorylation. These effects were rebalanced by silibinin. Considering several serine/threonine kinases reported to phosphorylate IRS-1 at Ser307, treatment with silibinin downregulated the phosphorylation of both c-Jun N-terminal kinase (JNK) and nuclear factor-κB kinase β (IKKβ), which was increased by palmitate in C2C12 myotubes mediating inflammatory status, whereas the phosphorylation of PKC-θ was not significantly modulated by silibinin. Collectively, the results indicated that silibinin prevented inhibition of the IRS-1/PI3K/Akt pathway, thus ameliorating palmitate-induced insulin resistance in C2C12 myotubes.

Vagotomy ameliorates islet morphofunction and body metabolic homeostasis in MSG-obese rats

The parasympathetic nervous system is important for β-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic β-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca2+ mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and β-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and β-cell area/pancreas section, respectively. Also, the β-cell number per β-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca2+ mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater β-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats.

Genetic purity certificate in seeds of hybrid maize using molecular markers

One of the main features that confer high quality to the seed is its genetic purity, in which one of the major causes of contamination is the self-pollination of the female parent. Up to date, there is no accurate and fast methods for detecting such contamination. Thus, this work was carried out to certify the genetic purity in seeds of hybrid maize using different biochemical and DNA-based markers. Two single-cross hybrids and their parental lines derived from the maize breeding program at UFLA were evaluated by isoenzymatic pattern of alcohol dehydrogenase (ADH), esterase (EST), acid phosphatase (ACP), glutamate-oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphoglucomate dehydrogenase (PGDH), catalase (CAT) and ß-glucosidade (ßGLU) and by microsatellites markers. The enzymatic systems that were able to distinguish the hybrids from their parental line were the catalase, the isocitrate dehydrogenase and the esterase. The esterase showed a Mendelian segregation pattern for UFLA 8/3 hybrid, that enables a safer genetic purity certificate. Microsatellites were able to differentiate the hybrid lines and the respective parental lines. Moreover, this technique was fast, precise and without environment effects. For microsatellites, the amplification pattern was identical when young leaves or seeds were used as DNA source. The possibility of using seeds as DNA source would accelerate and facilitate the role process of the genetic purity analysis.

Structural and Mechanistic Studies of Phosphoserine Aminotransferase

Phosphoserine aminotrasferase (PSAT: EC 2.6.1.52) is a vitamin B6-dependent enzyme and a member of the subgroup IV in the aminotransferase superfamily. Here, X-ray crystallography was used to determine the structure of PSAT from Bacillus alcalophilus with pyridoxamine 5′-phosphate (PMP) at high resolution (1.57 Å). In addition, analysis of active residues and their conformational changes was performed. The structure is of good quality as indicated, for example, by the last recorded Rwork and Rfree numbers (0.1331 and 0.1495, respectively). The enzyme was initially crystallized in the presence of substrate L-glutamate with the idea to produce the enzyme-substrate complex. However, the structure determination revealed no glutamate bound at the active site. Instead, the Schiff base between Lys196 and PLP appeared broken, resulting in the formation of PMP owing to the excess of the donor substrate used during co-crystallization. Structural comparison with the free PSAT enzyme and the PSAR-PSER complex showed that the aromatic ring of the co-factor remains in almost the same place in all structures. A flexible nearby loop in the active site was found in the same position as in the free PSAT structure while in the PSAT-PSER structure it moves inwards to interact with PSER. B-factors comparison in all three structures (PSAT-PMP complex, free PSAT, and PSAT-PSER complex) showed elevated loop flexibility in the absence of the substrate, indicating that loop flexibility plays an important role during substrate binding. The reported structure provides mechanistic details into the reaction mechanism of PSAT and may help in understanding better the role of various parts in the structure towards the design of novel compounds as potential disruptors of PSAT function. This may lead to the development of new drugs which could target the human and bacterial PSAT active site.

Does Gamma-aminobutyric acid function as a plant resistance mechanism against phytophagous insect activity? /

Gamma-aminobutyric acid (GAB A) is a ubiquitous non-protein amino acid synthesized via the decarboxylation of L-glutamate in a reaction catalyzed by the cytosolic enzyme L-glutamate decarboxylase (GAD). In animals it functions as an inhibitory neurotransmitter. In plants it accumulates rapidly in response to various stresses, but its function remains unclear. The hypothesis that GABA accumulation in leaf tissue may function as a plant resistance mechanism against phytophagous insect activity was investigated. GABA accumulation in response to mechanical stimulation, mechanical damage and insect activity was demonstrated. In wt tobacco (Nicotiana tabacum cv Samsun), mechanical stimulation or damage caused GABA to accumulate within 2 min from mean levels of 14 to 37 and 1~9 nmol g-l fresh weight (FW), respectively. In the transgenic tobacco strain CaMVGAD27c overexpressing Petunia GAD, the same treatments caused GABA to accumulate from 12 to 59 and 279 nmol g-l FW, respectively. In the transgenic tobacco strain CaMVGADilC 11 overexpressing Petunia GAD lacking an autoinhibitory domain, mechanical stimulation or damage caused GABA to accumulate from 180 to 309 and 630 nmol g-l FW, respectively. Ambulatory activity by tobacco budworm (TBW) larvae (Heliothis virescens) on leaves of CaMVGAD27c tobacco caused GABA to accumulate from 28 to 80 nmol g-l FW within 5 min. Ambulatory and leaf-rolling activity by oblique banded leaf roller (OBLR) larvae (Choristoneura rosaceana cv Harris) on wt soybean leaves (Glycine max cv Harovinton) caused GABA to accumulate from 60 to 1123 nmol g-l FW within 20 min. Increased GABA levels in leaf tissue were shown to affect phytophagous preference in TBW larvae presented with wt and transgenic tobacco leaves. When presented with leaves of Samsun wt and CaMVGAD27c plants, TBW larvae consumed more wt leaf tissue (640 ± 501 S.D. mm2 ) than transgenic leaf tissue (278 ± 338 S.D. mm2 ) nine times out of ten. When presented with leaves of Samsun wt and CaMVGAD~C11 plants, TBW larvae consumed more transgenic leaf tissue (1219 ± 1009 S.D. mm2 ) than wt leaf tissue (28 ± 31 S.D. mm2 ) ten times out of ten. These results indicate that: (1) ambulatory activity of insect larvae on leaves results in increased GABA levels, (2) transgenic tobacco leaves with increased capacity for GABA synthesis deter feeding, and (3) transgenic tobacco leaves with constitutively higher GABA levels stimulate feeding.

Effects of a hydroxy-cinnamoyl conjugate of spermidine in the neuromuscular junction of crayfish /

N'-coumaroyl spermidine (NlCSpd) is a plant derived chemical which is proposed to belong to a class of low molecular weight neuroactive substances called phenolic polyamines. NlCSpd is stnicturally similar to glutamate receptor blocking toxins found in certain spiders and wasps, such as JSTX-3 and NSTX-3 found in Nephila spiders. The goal of the present study was to determine if plant-derived phenolic polyamines act like other structurally related chemicals found in Arthropod venoms, such as JSTX-3, and whether they can be classified in the same pharmacological group as the spider and wasp toxins. A comparison was made to determine the relative potencies of various phenolic polyamines fi-om plants and insect venoms. This comparison was done by measuring the effect of various concentrations ofNlCSpd on the amplitude of excitatory postsynaptic potentials (EPSPs) elicited in muscle of the crayfish Proccanbarus clarkii. NlCSpd was also tested on L-glutamate induced potentials to determine if a postsynaptic component to sj^naptic block occurs. NlCSpd and an analogue with an a longer polyamine chain, NlCSpm, blocked EPSPs in a dose dependent manner, NlCSpd having an IC50 of lOOnM. NlCSpd also blocked L-glutamate induced potentials. The two main components of the NlCSpd molecule alone are insufficient for activity. NlCSpd acts postsynaptically by interfering with crayfish glutamatergic synaptic transmission, likely blocking glutamate receptors by interacting with the same site(s) as other phenolic polyamines. Certain moieties on the polyamines molecule are necessary for activity while others are not.

Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.