Tumor-Promoting Effects of Microrna-421/4-Aminobutyrate Aminotransferase Axis in Hepatocellular Carcinoma.

Background: MicroRNA-421 (miR-421) has been implicated in hepatocellular carcinoma (HCC), but its potential mechanism in HCC remains unclear. Objectives: The study aimed to study the potential mechanism of miR-421 in HCC which is necessary. Methods: The downstream target genes of miR-421 were screened in HCC tissues and cells using miDIP, Targetscan, and starBase databases. Differential analysis, survival analysis, and Pearson correlation analysis were performed between miR-421 and its downstream target genes. Quantitative reverse transcription polymerase chain reaction and western blot were used to assay RNA and protein levels of 4-aminobutyrate aminotransferase (ABAT) and epithelial-mesenchymal transition (EMT)-related proteins. Cell-based assays, including CCK-8, wound healing, transwell, flow cytometry, and metabolic measurements, were implemented to assess proliferation, migration, invasion, cell cycle, and apoptosis of HCC cells with different treatments. Dual-luciferase assay was utilized to detect the targeting relationship between miR-421 and ABAT. Results: miR-421 level was elevated in HCC tissues and cells, and low miR-421 expression hindered phenotype progression of HCC cells. ABAT was identified as a direct target of miR-421 in HCC cells, and miR-421 could inhibit ABAT expression. Rescue assay revealed that miR-421 promoted HCC cell tumorigenesis progress and affected cell metabolic remodeling through down-regulating ABAT. Conclusion: The miR-421/ABAT regulatory axis promoted HCC cell tumorigenesis progress, highlighting its potential as a therapeutic target for HCC.

Positive Predictive Value Surfaces as a Complementary Tool to Assess the Performance of Virtual Screening Methods.

BACKGROUND: Since their introduction in the virtual screening field, Receiver Operating Characteristic (ROC) curve-derived metrics have been widely used for benchmarking of computational methods and algorithms intended for virtual screening applications. Whereas in classification problems, the ratio between sensitivity and specificity for a given score value is very informative, a practical concern in virtual screening campaigns is to predict the actual probability that a predicted hit will prove truly active when submitted to experimental testing (in other words, the Positive Predictive Value - PPV). Estimation of such probability is however, obstructed due to its dependency on the yield of actives of the screened library, which cannot be known a priori. OBJECTIVE: To explore the use of PPV surfaces derived from simulated ranking experiments (retrospective virtual screening) as a complementary tool to ROC curves, for both benchmarking and optimization of score cutoff values. METHODS: The utility of the proposed approach is assessed in retrospective virtual screening experiments with four datasets used to infer QSAR classifiers: inhibitors of Trypanosoma cruzi trypanothione synthetase; inhibitors of Trypanosoma brucei N-myristoyltransferase; inhibitors of GABA transaminase and anticonvulsant activity in the 6 Hz seizure model. RESULTS: Besides illustrating the utility of PPV surfaces to compare the performance of machine learning models for virtual screening applications and to select an adequate score threshold, our results also suggest that ensemble learning provides models with better predictivity and more robust behavior. CONCLUSION: PPV surfaces are valuable tools to assess virtual screening tools and choose score thresholds to be applied in prospective in silico screens. Ensemble learning approaches seem to consistently lead to improved predictivity and robustness.

QSAR and Molecular Docking Studies of the Inhibitory Activity of Novel Heterocyclic GABA Analogues over GABA-AT.

We have previously reported the synthesis, in vitro and in silico activities of new GABA analogues as inhibitors of the GABA-AT enzyme from Pseudomonas fluorescens, where the nitrogen atom at the γ-position is embedded in heterocyclic scaffolds. With the goal of finding more potent inhibitors, we now report the synthesis of a new set of GABA analogues with a broader variation of heterocyclic scaffolds at the γ-position such as thiazolidines, methyl-substituted piperidines, morpholine and thiomorpholine and determined their inhibitory potential over the GABA-AT enzyme from Pseudomonas fluorescens. These structural modifications led to compound 9b which showed a 73% inhibition against this enzyme. In vivo studies with PTZ-induced seizures on male CD1 mice show that compound 9b has a neuroprotective effect at a 0.50 mmole/kg dose. A QSAR study was carried out to find the molecular descriptors associated with the structural changes in the GABA scaffold to explain their inhibitory activity against GABA-AT. Employing 3D molecular descriptors allowed us to propose the GABA analogues enantiomeric active form. To evaluate the interaction with Pseudomonas fluorescens and human GABA-AT by molecular docking, the constructions of homology models was carried out. From these calculations, 9b showed a strong interaction with both GABA-AT enzymes in agreement with experimental results and the QSAR model, which indicates that bulky ligands tend to be the better inhibitors especially those with a sulfur atom on their structure.

Novel-Substituted Heterocyclic GABA Analogues. Enzymatic Activity against the GABA-AT Enzyme from Pseudomonas fluorescens and In Silico Molecular Modeling.

γ-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system, and a deficiency of GABA is associated with serious neurological disorders. Due to its low lipophilicity, there has been an intensive search for new molecules with increased lipophilicity to cross the blood-brain barrier to raise GABA concentrations. We have designed and evaluated in vitro and in silico some new analogues of GABA, where the nitrogen atom at the γ-position is embedded in heterocyclic scaffolds and determined their inhibitory potential over the GABA-AT enzyme from Pseudomonas fluorescens. These modifications lead to compounds with inhibitory activity as it occurs with compounds 18a and 19a. The construction of Pseudomonas fluorescens and human GABA-AT models were carried out by homology modeling. Docking assays were done for these compounds over the GABA-AT enzyme models where 19a showed a strong interaction with both GABA-AT enzymes.

Genotoxic and mutagenic effects of vigabatrin, a γ-aminobutyric acid transaminase inhibitor, in Wistar rats submitted to rotarod task.

Vigabatrin (VGB) is an antiepileptic drug thatincreases brain γ-aminobutyric acid (GABA) levels through irreversible inhibition of GABA transaminase. The aim of this study was to evaluate neurotoxicological effects of VGB measuring motor activity and genotoxic and mutagenic effects after a single and repeated administration. Male Wistar rats received saline, VGB 50, 100, or 250 mg/kg by gavage for acute and subchronic (14 days) treatments and evaluated in the rotarod task. Genotoxicity was evaluated using the alkaline version of the comet assay in samples of blood, liver, hippocampus, and brain cortex after both treatments. Mutagenicity was evaluated using the micronucleus test in bone marrow of the same animals that received subchronic treatment. The groups treated with VGB showed similar performance in rotarod compared with the saline group. Regarding the acute treatment, it was observed that only higher VGB doses induced DNA damage in blood and hippocampus. After the subchronic treatment, VGB did not show genotoxic or mutagenic effects. In brief, VGB did not impair motor activities in rats after acute and subchronic treatments. It showed a repairable genotoxic potential in the central nervous system since genotoxicity was observed in the acute treatment group.

Assessment of in vitro pharmacological effect of Neotropical Piperaceae in GABAergic bioassays in relation to plants traditionally used for folk illness by the Yanesha (Peru).

ETHNOPHARMACOLOGICAL RELEVANCE: A previous pilot ethnobotanical and ethnopharmacological study with the Q'echi׳ Maya identified the family Piperaceae, as an important taxonomic group traditionally used for the treatment of epileptic and culture-bound anxiety disorders and possessing activity in the GABA system. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae were collected including 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an indigenous Amazonian group. MATERIALS AND METHODS: Two high throughput bioassays were used to quantify the in vitro activity of botanical extracts on the GABA system. RESULTS: Plant extracts demonstrated moderate to high affinity to the γ-aminobutyric acid benzodiazepine (GABA-BZD) receptor. In addition, extracts demonstrated low to moderate activity in the inhibition of the GABA-transaminase, with select plants exhibiting significant activity. Plants indicated by the Yanesha showed comparable activity to the other Piperaceae plants collected. Piper cremii was the most active plant in the GABA-BZD receptor assay, and Drymaria cordata (Caryophyllaceae) in the GABA-T assay. CONCLUSION: The study provides evidence that there is a pharmacological basis behind the use of plants in the treatment of susto and mal aire in both Central and South America, and we propose that the possible mechanism of action includes an interaction with the GABA-T enzyme and/or the GABAA-BZD receptor.

Synchronization by food access modifies the daily variations in expression and activity of liver GABA transaminase.

Daytime restricted feeding (DRF) is an experimental protocol that influences the circadian timing system and underlies the expression of a biological clock known as the food entrained oscillator (FEO). Liver is the organ that reacts most rapidly to food restriction by adjusting the functional relationship between the molecular circadian clock and the metabolic networks. γ-Aminobutyric acid (GABA) is a signaling molecule in the liver, and able to modulate the cell cycle and apoptosis. This study was aimed at characterizing the expression and activity of the mostly mitochondrial enzyme GABA transaminase (GABA-T) during DRF/FEO expression. We found that DRF promotes a sustained increase of GABA-T in the liver homogenate and mitochondrial fraction throughout the entire day-night cycle. The higher amount of GABA-T promoted by DRF was not associated to changes in GABA-T mRNA or GABA-T activity. The GABA-T activity in the mitochondrial fraction even tended to decrease during the light period. We concluded that DRF influences the daily variations of GABA-T mRNA levels, stability, and catalytic activity of GABA-T. These data suggest that the liver GABAergic system responds to a metabolic challenge such as DRF and the concomitant appearance of the FEO.

Common features and differences in the expression of the three genes forming the UGA regulon in Saccharomyces cerevisiae.

The three genes that form the UGA regulon in Saccharomyces cerevisiae are responsible for the transport and degradation of γ-aminobutyric acid (GABA) in this organism. Despite the differences in the sequence of their promoters, these genes similarly respond to GABA stimuli. The expression of UGA1, UGA2 and UGA4 depends on GABA induction and nitrogen catabolite repression (NCR). The induction of these genes requires the action of at least two positive proteins, the specific Uga3 and the pleiotropic Uga35/Dal81 transcription factors. Here we show that all the members of the UGA regulon, as was already demonstrated for UGA4, are negatively regulated by extracellular amino acids through the SPS amino acid sensor. We also show that this negative effect is caused by a low availability of Uga35/Dal81 transcription factor and that Leu3 transcription factor negatively regulates UGA4 and UGA1 expression but it does not affect UGA2 expression.

Ethnopharmacology of Q'eqchi' Maya antiepileptic and anxiolytic plants: effects on the GABAergic system.

ETHNOPHARMACOLOGICAL RELEVANCE: The Q'eqchi' Maya possess a large selection of plants to treat neurological disorders, including epilepsy and susto (fright), a culture-bound illness related to anxiety disorders. AIM OF THE STUDY: To investigate the activity of antiepileptic and anxiolytic plants in the GABAergic system, and determine if there is a pharmacological basis for plant selection. MATERIALS AND METHODS: Ethanol extracts of 34 plants were tested in vitro for their ability to inhibit GABA-transaminase (GABA-T) or bind to the GABA(A)-benzodiazepine (BZD) receptor, two principal drug targets in epilepsy and anxiety. Pharmacological activity was correlated with relative frequency of use, based on informant consensus. RESULTS: Ten plants showed greater than 50% GABA-T inhibition at 1mg/ml, while 23 showed greater than 50% binding to the GABA(A)-BZD receptor at 250 microg/ml. Piperaceae, Adiantaceae and Acanthaceae families were highly represented and active in both assays. There was a significant positive correlation between GABA-T inhibition and relative frequency of use for epilepsy, and an even stronger correlation between GABA(A) binding and relative frequency of use for susto (fright). CONCLUSIONS: Clearly, Q'eqchi' traditional knowledge of antiepileptic and anxiolytic plants is associated with the use of pharmacologically active plants. Based on the evidence, it is suggested that the mechanism of action for some traditionally used plants may be mediated through the GABAergic system.

GABAergic drugs inhibit amphetamine-induced distractibility in the rat.

Drugs facilitating GABAergic neurotransmission have been reported to block some behavioral actions of dopaminergic stimulation but not others. The present experiments were performed with the purpose to extend the range of behaviors in which the interaction between GABA and dopamine have been studied. The ability of the GABAB agonist baclofen and the GABA transaminase inhibitor sodium valproate to block the enhanced distractibility produced by amphetamine was evaluated in a procedure especially designed for analyzing drugs' effects on distractibility. Briefly, rats were trained to traverse a straight runway with a sucrose solution as reinforcement. Once the response had been acquired, an additional runway ending in an empty box was connected. The time spent investigating this additional runway is the measure of distractibility. Male rats treated with amphetamine, 1 mg/kg, displayed an increase of the time spent in the additional runway. Baclofen, 2.5 and 5 mg/kg, and sodium valproate, 100 and 200 mg/kg, had no effect on distraction behavior when administered alone. However, when these drugs were administered together with amphetamine, 1 mg/kg, they completely inhibited the effects of the stimulant on distractibility. These data show that distractibility is similar to discrimination learning with regard to the capacity of GABAergic drugs to block the effects of dopaminergic stimulation. It is different from locomotor activity, however, where GABAergic drugs are ineffective in this respect.

Vigabatrin como droga agregada en 20 niños con epilepsia de difícil control: evaluación a los 3 y 6 meses de tratamiento / Add-on vigabatrin therapy in 20 children with intractable epilepsy

Con el propósito de demostrar la eficacia y tolerabilidad del vigabatrin en pacientes pediátricos, hemos utilizado esta droga en 20 niños con síndromes epilépticos refractarios al tratamiento tradicional, como droga agregada y sin modificar el esquema terapéutico anterior. El 47 por ciento de los pacientes demostró una muy buena respuesta clínica (reducción del número de crisis>75 por ciento ), y el 16 por ciento una buena respuesta (reducción del 50 al 74 por ciento ). No se observaron cambios significativos en el 21 por ciento y el 16 por ciento restante evidenció un incremento en el número de crisis. El vigabatrin mostró mayor eficacia en los pacientes con epilepsias parciales criptogénicas y sintomáticas. Los resultados presentados en este estudio permiten considerar al vigabatrin como una alternativa útil en el tratamiento de los síndromes epilépticos refractarios en la infancia (AU)

Vigabatrin como droga agregada en 20 niños con epilepsia de difícil control: evaluación a los 3 y 6 meses de tratamiento / Add-on vigabatrin therapy in 20 children with intractable epilepsy

Con el propósito de demostrar la eficacia y tolerabilidad del vigabatrin en pacientes pediátricos, hemos utilizado esta droga en 20 niños con síndromes epilépticos refractarios al tratamiento tradicional, como droga agregada y sin modificar el esquema terapéutico anterior. El 47 por ciento de los pacientes demostró una muy buena respuesta clínica (reducción del número de crisis>75 por ciento ), y el 16 por ciento una buena respuesta (reducción del 50 al 74 por ciento ). No se observaron cambios significativos en el 21 por ciento y el 16 por ciento restante evidenció un incremento en el número de crisis. El vigabatrin mostró mayor eficacia en los pacientes con epilepsias parciales criptogénicas y sintomáticas. Los resultados presentados en este estudio permiten considerar al vigabatrin como una alternativa útil en el tratamiento de los síndromes epilépticos refractarios en la infancia

Changes in the effect of gamma-aminobutyric acid on prolactin secretion during sexual maturation in female rats.

To evaluate the effects of the gamma-aminobutyric acid (GABA)ergic system on PRL secretion during sexual development in female rats, aminooxyacetic acid (an inhibitor of GABA-transaminase which increases the hypothalamic GABA concentration) was administered to 12- to 16- and 30-day-old female rats. The increased GABA level in the hypothalamus was accompanied by a significant increase in the serum PRL concentration in rats 16 days of age and a decrease in the serum concentration of the hormone in 30-day-old rats. No changes in PRL levels were observed at 12 days of age. Muscimol, a GABA-A receptor agonist, increased serum PRL concentrations in 16-day-old rats. By contrast, in rats 30 days of age, muscimol induced a significant decrease in PRL levels. On the other hand, the administration of baclofen, a GABA-B receptor agonist, induced a pattern of PRL modification similar to that observed with muscimol, i.e. stimulation in rats 16 days of age and inhibition in 30-day-old rats. These findings indicate that both the stimulatory and inhibitory effects of the GABAergic system on PRL secretion during sexual maturation are mediated by GABA-A and GABA-B receptors. The present results show that during sexual maturation there is a qualitative modification of the effect of the GABAergic system on PRL secretion. These findings could be related to the complex neuroendocrine mechanisms involved in the onset of puberty in the female rat.

Sexual behavior and copulatory thrusting patterns in male rabbits treated with GABA transaminase inhibitors.

The effects of enhanced central nervous system GABA levels on sexual behavior and copulatory pelvic thrusting were evaluated in male New Zealand white rabbits. The GABA transaminase inhibitors sodium valproate and gamma-acetylen GABA (GAG), in doses of 100 and 200 mg/kg and 50 and 100 mg/kg, respectively, were intraperitoneally administered and sexual behavior recorded at several intervals after drug administration. At the same time, copulatory thrusting was registered using a polygraphic technique. Tests for gross motor functions were also performed. None of the drugs had any effect in these latter tests. Sodium valproate, in a dose of 100 mg/kg, had a slight inhibitory effect on sexual behavior at 280 min postinjection. A dose of 200 mg/kg inhibited sexual activity already 15 min postinjection, and the effect lasted for at least 280 min. GAG, 100 mg/k, inhibited mounting behavior at 8 h postinjection, and ejaculation was reduced from 2 to at least 8 h postinjection. Copulatory thrusting patterns were not affected by the drug treatments. These data suggest that increased GABAergic activity reduces sexual arousal in the rabbit. GABA does not seem to be critically involved in the regulation of the motor patterns underlying pelvic thrusting. There are important quantitative and qualitative differences between rats and rabbits with regard to the actions of GABA transaminase inhibitors upon sexual functions.

Effects of serotonin on the hypothalamic-pituitary GABAergic system.

The effects of serotonin (5-HT) and its precursor, 5-hydroxytryptophan (5-HTP) on the GABAergic system in the mediobasal hypothalamus (MBH) and the anterior pituitary were studied. The IP administration of 5-HTP produced a transient increase (only at 45 min after the injection) in glutamate decarboxylase activity (GAD) of MBH and in GABA concentration in anterior pituitary. Besides, 5-HTP administration increased the in vitro evoked GABA release from MBH. The administration of 5-HTP to hypophysectomized rats partially reversed the inhibitory effects of hypophysectomy on GABA concentration in MBH. We also examined the direct effect of 5-HT on some parameters on the hypothalamic GABAergic system. The presence of 5-HT in the incubation medium increased GAD activity and evoked GABA release from MBH. These observations indicate that the serotoninergic stimulation of the hypothalamic GABAergic system could be a direct effect which may, at least partially, be independent of the feedback mechanism induced by prolactin on the GABAergic neurons. The serotoninergic increase of prolactin secretion could be accomplished through stimulation of the hypothalamic GABAergic transmission.

GABAergic modulation of yawning behavior.

The hypothetical modulation by GABAergic neurons of yawning behavior in the rat was explored with GABA-active drugs. Gamma-acetylenic-GABA, a specific inhibitor of GABA-T, increases yawning frequency when injected at a dose of 7 mg/kg. Baclofen, a GABAB agonist (3 mg/kg), inhibits yawning completely; GABA antagonists, bicuculline and picrotoxin, at subconvulsant doses, also decrease yawning. All drugs were injected intraperitoneally with the exception of apomorphine, which was injected subcutaneously. It is suggested that GABAB receptors play a role in yawning behavior by modulating ACh release, and that GABAA receptors may modify yawning frequency by modulating inhibitory influences on ACh neurons.

Acute stress and GABAergic function in the rat brain.

1. The function of the gamma-aminobutyric acid (GABA)-ergic system in certain areas of the rat brain was investigated after acute (5 min) exposure to immobilization stress. 2. The activities of glutamate decarboxylase and GABA-transaminase, GABA concentrations, GABA turnover in vivo and uptake of [3H]-GABA were measured. 3. After 5 min of immobilization stress, GABA concentrations and [3H]-GABA uptake were reduced, and GABA turnover stimulated in the olfactory bulbs. In contrast the uptake of [3H]-GABA was increased in the corpus striatum after 5 min of immobilization stress. 4. None of the parameters measured was significantly altered by acute immobilization stress in the frontal cortex, hippocampus or medio-basal hypothalamus. 5. These findings show that the olfactory bulbs and the corpus striatum are sensitive to the effects of acute stress. Since GABA in the olfactory bulbs is involved in the development of aggression and increased emotional state, it follows that neurochemical changes induced by acute stress might underlie some behavioural manifestations observed after stress.

A comparison of GABAergic influences on the analgesic responses to morphine and pentazocine.

1. The interactions of three GABAergic compounds, gamma-acetylenic GABA, gamma-vinyl GABA and ethylenediamine with the analgesic effects of morphine and pentazocine were examined in mice using the hot plate and tail immersion tests. 2. A significant increase in reaction time induced by morphine was noted in the tail immersion test after pretreatment with the drugs acting through GABA functions. 3. The inhibitors of GABA transaminase, gamma-acetylenic GABA and gamma-vinyl GABA, and the GABAmimetic ethylenediamine did not significantly change the analgesic action induced by pentazocine. 4. In the hot plate test the three GABAergic compounds antagonized the analgesic effects of pentazocine in contraposition with previous results indicating that morphine-induced analgesia is increased by pretreatment with those agents. 5. These findings suggest that GABAergic and opiopeptidergic systems are interconnected through mu receptors, whereas the kappa opiate systems seem to be unrelated to GABA functions.

Estriol affects prolactin and LH secretion in rats.

The effects of estriol on serum prolactin (PRL) and LH levels, on the pituitary response to TRH and LHRH and on the synthesis and release of PRL from the anterior pituitary gland were investigated in female rats. The increase of serum PRL levels after estradiol administration was found to be associated with an increase of glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T) in the hypothalamus. Thus, a study was carried out on the effects of estradiol and estriol on PRL secretion and on GAD, GABA-T and gamma-amino butyric acid (GABA) in the hypothalamus and the anterior pituitary. Under basal and TRH-stimulated conditions, estriol increased serum PRL levels, decreased basal serum LH levels, and increased the response to LHRH, in terms of LH release. Estradiol and estriol increased the synthesis and release of 3H-PRL from hemipituitary glands in incubations of pretreated animals. Both estrogens induced hyperprolactinemia, concomitantly with an increase of hypothalamic GAD and GABA-T activity. Estriol increased hypothalamic GABA concentration, but did not modify GABA concentration in the pituitary glands. Our results show that estriol, at relatively high doses, seems to be active in increasing PRL synthesis and release and in decreasing serum LH levels; it can also modify pituitary response to TRH and LHRH stimulation.