Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

Modulation of GABAergic neurotransmission in the brain by dipeptides

The effects of endogenous and synthetic peptides containing GABA or its analogues on the GABA/benzodiazepine/chloride ionophore, complex, GABA_B receptor, Cl fluxes, GABA release and GABA uptake were studied using synaptic membranes, crude synaptoneurosomal preparations and slices prepared from the rat and mouse brain. The sodium-independent binding of GABA was strongly inhibited by GABA-histidine, followed by -glutamyl-homotaurine, GABA-glycine and -glutamyl-GABA. The binding of diazepam was slightly enhanced by the same peptides. The peptides alone had no effect on the chloride fluxes, but GABA-histidine, -glutamyl-GABA and GABA-glycine enhanced while -glutamyl-homotaurine and GABA-taurine inhibited GABA-stimulated chloride uptake. GABA-histidine was the most effective displacer of baclofen binding, but -glutamyl-homotaurine was entirely ineffective. The uptake of GABA was markedly inhibited in synaptosomal preparations by GABA-histidine, while all other peptides were less effective. -Glutamyl-taurine attenuated but -glutamyl-homotaurine and GABA-glycine enhanced the potassium-stimulated release of GABA. The present actions of GABA-histidine in vitro may be of significance for GABAergic neurotransmission in vivo.     

Evaluation of the prevalence and economic burden of adverse drug reactions presenting to the medical emergency department of a tertiary referral centre: a prospective study

Background  

Adverse drug reactions (ADRs) are now recognized as an important cause of hospital admissions, with a proportion ranging from 0.9–7.9%. They also constitute a significant economic burden. We thus aimed at determining the prevalence and the economic burden of ADRs presenting to Medical Emergency Department (ED) of a tertiary referral center in India     

Characterization of the two-component monooxygenase system AlnT/AlnH reveals early timing of quinone formation in alnumycin biosynthesis

Alnumycin A is an aromatic polyketide with a strong resemblance to related benzoisochromanequinone (BIQ) antibiotics, such as the model antibiotic actinorhodin. One intriguing difference between these metabolites is that the positions of the benzene and quinone rings are reversed in alnumycin A in comparison to the BIQ polyketides. In this paper we demonstrate that inactivation of either the monooxygenase alnT gene or the flavin reductase alnH gene results in the accumulation of a novel nonquinoid metabolite, thalnumycin A (ThA), in the culture medium. Additionally, two other previously characterized metabolites, K1115 A and 1,6-dihydroxy-8-propylanthraquinone (DHPA), were identified, which had oxidized into quinones putatively nonenzymatically at the incorrect position in the central ring. None of the compounds isolated contained correctly formed pyran rings, which suggests that on the alnumycin pathway quinone biosynthesis occurs prior to third ring cyclization. The regiochemistry of the two-component monooxygenase system AlnT/AlnH was finally confirmed in vitro by using ThA, FMN, and NADH in enzymatic synthesis, where the reaction product, thalnumycin B (ThB), was verified to contain the expected p-hydroquinone structure in the lateral ring.     

High dexamethasone concentration prevents stimulatory effects of TNF-alpha and LPS on IL-6 secretion from the precursors of human muscle regeneration

A frequent finding in patients surviving critical illness myopathy is chronic muscle dysfunction. Its pathogenesis is mostly unknown; one explanation could be that muscle regeneration, which normally follows myopathy, is insufficient in these patients because of a high glucocorticoid level in their blood. Glucocorticoids can prevent stimulatory effects of proinflammatory factors on the interleukin (IL)-6 secretion, diminishing in this way the autocrine and paracrine IL-6 actions known to stimulate proliferation at the earliest, myoblast stage of muscle formation. To test this hypothesis, we compared the effects of major proinflammatory agents [tumor necrosis factor (TNF)-alpha and endotoxin lipopolysaccharide (LPS)] on the IL-6 secretion from the muscle precursors and then studied the influence of dexamethasone (Dex) on these effects. Mononuclear myoblasts, which still proliferate, were compared with myotubes in which this capacity is already lost. For correct interpretation of results, cultures were examined for putative apoptosis and necrosis. We found that constitutive secretion of IL-6 did not differ significantly between myoblasts and myotubes; however, the TNF-alpha- and LPS-stimulated IL-6 release was more pronounced (P < 0.001) in myoblasts. Dex, applied at the 0.1-100 nM concentration range, prevented constitutive and TNF-alpha- and LPS-stimulated IL-6 release at both developmental stages but only at high concentration (P < 0.01). Although there are still missing links to it, our results support the concept that high concentrations of glucocorticoids, met in critically ill patients, prevent TNF-alpha- and LPS-stimulated IL-6 secretion. This results in reduced IL-6-mediated myoblast proliferation, leading to the reduced final mass of the regenerated muscle.     

Matrix association of latent TGF‐beta binding protein‐2 (LTBP‐2) is dependent on fibrillin‐1

The components of the extracellular matrix (ECM) and their differential expression patterns play important roles in tissue formation. The deposition of latent TGF‐β binding proteins (LTBPs) to the ECM exhibit distinct distribution profiles. We have analyzed here the temporal and spatial ECM association of latent TGF‐β binding protein LTBP‐2 in cultured human embryonic lung fibroblasts. We found that LTBP‐2 was not assembled to the ECM until by confluency of cultures following the deposition of fibronectin (FN) and fibrillin‐1. In 5‐day‐old cultures LTBP‐2 was rapidly secreted from cells and it subsequently associated with the ECM as shown by metabolic labeling and immunoprecipitation. LTBP‐2 colocalized transiently with fibronectin and failed to assemble to the ECM of FN deficient mouse fibroblasts. Analysis of different cultured human cell lines revealed partial colocalization of LTBP‐2 and fibrillin‐1 in the ECM of fibroblasts, MG‐63 osteosarcoma cells and human vascular endothelial cells. Silencing of fibrillin‐1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP‐2. Current results suggest that LTBP‐2 is not an element of the provisional ECM of fibroblasts but is more likely a component of more mature ECM and indicate that matrix association of LTBP‐2 depends on a pre‐formed fibrillin‐1 network. J. Cell. Physiol. 221: 586–593, 2009. © 2009 Wiley‐Liss, Inc.     

Osteoarthritis and nutrition. From nutraceuticals to functional foods: a systematic review of the scientific evidence

The scientific and medical community remains skeptical regarding the efficacy of nutrition for osteoarthritis despite their broad acceptation by patients. In this context, this paper systematically reviews human clinical trials evaluating the effects of nutritional compounds on osteoarthritis. We searched the Medline, Embase, and Biosis databases from their inception to September 2005 using the terms random, double-blind method, trial, study, placebo, and osteoarthritis. We selected all peer-reviewed articles reporting the results of randomised human clinical trials (RCTs) in osteoarthritis that investigated the effects of oral interventions based on natural molecules. Studies on glucosamine and chondroitin sulfate were excluded. The quality of the RCTs was assessed with an osteoarthritic-specific standardised set of 12 criteria and a validated instrument. A best-evidence synthesis was used to categorise the scientific evidence behind each nutritional compound as good, moderate, or limited. A summary of the most relevant in vitro and animal studies is used to shed light on the potential mechanisms of action. Inclusion criteria were met by 53 RCTs out of the 2,026 identified studies. Good evidence was found for avocado soybean unsaponifiables. Moderate evidence was found for methylsulfonylmethane and SKI306X, a cocktail of plant extracts. Limited evidence was found for the Chinese plant extract Duhuo Jisheng Wan, cetyl myristoleate, lipids from green-lipped mussels, and plant extracts from Harpagophytum procumbens. Overall, scientific evidence exists for some specific nutritional interventions to provide symptom relief to osteoarthritic patients. It remains to be investigated whether nutritional compounds can have structure-modifying effects.     

Enhanced taurine release in cultured cerebellar granule cells in cell-damaging conditions

Summary The release of taurine from cultured cerebellar granule neurons was studied in different cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and in the presence of free radicals. The effects of both ionotropic and metabotropic glutamate receptor agonists on the release were likewise investigated. The release of [³H]taurine from the glutamatergic granule cells was increased by K⁺ (50mM) and veratridine (0.1 mM), the effect of veratridine being the greater. Hypoxia and ischemia produced an initial increase in release compared to normoxia but resulted in a diminished response to K. Hypoglycemia, oxidative stress and free radicals enhanced taurine release, and subsequent K^– treatment exhibited a correspondingly greater stimulation. A common feature of taurine release in all the bove conditions was a slow response to the stimulus evoked by K⁺ and particularly to that evoked by veratridine. All ionotropic glutamate receptor agonists potentiated taurine release, but only the action of kainate seemed to be receptor-mediated. Metabotropic receptor agonists of group I slightly stimulated the release. The prolonged taurine release seen in both normoxia and cell-damaging conditions may be of importance in maintaining homeostasis in the cerebellum and reducing excitability for a longer period than other neuroprotective mechanisms.Abbreviations AIDA (RS)-1-aminoindan-1,5-dicarboxylate - AMPA 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate - CNOX 6-cyano-7-nitroquinoxaline-2,3-dione - DCG IV (2S,2R,3R)-2-(2,3-dicarboxycyclo-propyl)glycine - DHPG (S)-3,5-dihydroxyphenylglycine - EGLU (2S)-2-ethylglutamate - L-AP3 L(+)-2-amino-3-phosphonopropionate - L-AP4 L(+)-2-amino-4-phosphonobutyrate - L-SOP o-phospho-l-serine - NBOX 6-nitro-7-sulphamoyl[f]quinoxaline-2,3-dione - NMDA n-methyl-d-aspartate - trans-ACPD (1S,3S)-1-aminocyclopentane-1,3-dicarboxylate     

Effect of Sulpiride on the Amphetamine-Induced Changes in Extracellular Dopamine,DOPAC, and Hydroxyl Radical Generation in the Rat Striatum

The neurotoxic effects of psychostimulants are mediated by several mechanisms, which together lead to neuronal damage. These mechanisms include an increase in the extracellular content of dopamine, stimulation of dopamine oxidation, accumulation of extracellular glutamate, and an increase in body temperature. In the present study, the dopamine receptor antagonist sulpiride proved able to prevent the delayed loss of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) and depressed the gradual generation of hydroxyl radicals induced in the rat striatum by D-amphetamine. However, sulpiride at a dose of 75 mg/kg × 2, coadministered with D-amphetamine (7.5 mg/kg × 4), potentiated the increase in extracellular dopamine and initially slightly enhanced D-amphetamine-induced stereotypy. The gradual increase in hydroxyl radical generation predicts the depletion of dopamine and DOPAC in the rat striatum after D-amphetamine administration, but the increase in extracellular dopamine is not a pivotal factor in the enhanced production of hydroxyl radicals.     

Cyclic AMP-mediated regulation of striatal glutamate release: interactions of presynaptic ligand- and voltage-gated ion channels and G-protein-coupled receptors

The presynaptic regulation of striatal glutamate transmission was investigated using D-[3H]aspartate and mouse striatal slices. Functional changes in voltage-dependent and glutamate receptor-gated ion channels were elicited by pharmacologically modifying intracellular cyclic AMP formation via G-protein-coupled receptor stimulation. The kainate (KA)-evoked release was potentiated by the stimulatory G-protein (G(s))-coupled beta-adrenoceptor agonist isoproterenol (ISO) in a concentration-dependent manner. This effect was mimicked by the specific calmodulin (CaM) antagonists trifluoperazine and calmidazolium. Tetrodotoxin (TTX), a blocker of Na(+) channels, did not affect the basal release but inhibited to the same degree the releases evoked by kainate alone and by kainate and isoproterenol together. Vinpocetine, a blocker of voltage-dependent Na(+) channels, did not alter the basal or the evoked release. The Na(+) channel activator veratridine enhanced the basal release in a concentration-dependent manner and isoproterenol attenuated this effect. The opposite effects of isoproterenol on the kainate- and veratridine-evoked releases may reflect prevention of the cyclic AMP-protein kinase A (PKA) phosphorylation cascade in striatal glutamatergic signal transduction. In addition, the calmidazolium-induced potentiation of kainate-evoked release was thwarted by LY354740 and L-2-amino-4-phosphonobutanoate, agonists of the inhibitory G-protein (G(i))-coupled metabotropic group II and III glutamate receptors (mGluRs). Vinpocetine, which inhibits the CaM-dependent phosphodiesterase (PDE1), was likewise inhibitory. In turn, selective agonists and antagonists of the G(q)-protein-coupled group I mGluRs and (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylate (AIDA), which modulate the intracellular Ca(2+) levels, did not alter the kainate-evoked release.The beta-adrenoceptor-mediated cyclic AMP accumulation seems to downregulate Na(+) channels but to enhance glutamate release by means of upregulation of kainate receptors. This regulation of presynaptic ligand- and voltage-gated ion channels is affected by the cAMP-protein kinase A-dependent phosphorylation cascade and controlled by G(i)-protein-coupled mGluRs.