Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds

Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.     

Critical enzymes involved in endocannabinoid metabolism

Investigations of the pathways involved in the metabolism of endocannabinoids have grown exponentially in recent years following the discovery of cannabinoid receptors (CB) and their endogenous ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The in vivo biosynthesis of AEA has been shown to occur through several pathways mediated by N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), a secretory PLA(2) and PLC. 2-AG, a second endocannabinoid is generated through the action of selective enzymes such as phosphatidic acid phsophohydrolase, diacylglycerol lipase (DAGL), phosphoinositide-specific PLC (PI-PLC) and lyso-PLC. A putative membrane transporter or facilitated diffusion is involved in the cellular uptake or release of endocannabinoids. AEA is metabolized by fatty acid amidohydrolase (FAAH) and 2-AG is metabolized by both FAAH and monoacylglycerol lipase (MAGL). The author presents an integrative overview of current research on the enzymes involved in the metabolism of endocannabinoids and discusses possible therapeutic interventions for various diseases, including addiction.     

Acute ethanol suppresses glutamatergic neurotransmission through endocannabinoids in hippocampal neurons

Ethanol exposure during fetal development is a leading cause of long-term cognitive impairments. Studies suggest that ethanol exposure have deleterious effects on the hippocampus, a brain region that is important for learning and memory. Ethanol exerts its effects, in part, via alterations in glutamatergic neurotransmission, which is critical for the maturation of neuronal circuits during development. The current literature strongly supports the growing evidence that ethanol inhibits glutamate release in the neonatal CA1 hippocampal region. However, the exact molecular mechanism responsible for this effect is not well understood. In this study, we show that ethanol enhances endocannabinoid (EC) levels in cultured hippocampal neurons, possibly through calcium pathways. Acute ethanol depresses miniature post-synaptic current (mEPSC) frequencies without affecting their amplitude. This suggests that ethanol inhibits glutamate release. The CB1 receptors (CB1Rs) present on pre-synaptic neurons are not altered by acute ethanol. The CB1R antagonist SR 141716A reverses ethanol-induced depression of mEPSC frequency. Drugs that are known to enhance the in vivo function of ECs occlude ethanol effects on mEPSC frequency. Chelation of post-synaptic calcium by EGTA antagonizes ethanol-induced depression of mEPSC frequency. The activation of CB1R with the selective agonist WIN55,212-2 also suppresses the mEPSC frequency. This WIN55,212-2 effect is similar to the ethanol effects and is reversed by SR141716A. In addition, tetani-induced excitatory post-synaptic currents (EPSCs) are depressed by acute ethanol. SR141716A significantly reverses ethanol effects on evoked EPSC amplitude in a dual recording preparation. These observations, taken together, suggest the participation of ECs as retrograde messengers in the ethanol-induced depression of synaptic activities.     

Isolation and characterization of "Reprotoxin", a novel protein complex from Daboia russelii snake venom

In snake venoms, non-covalent protein-protein interaction leads to protein complexes with synergistic and, at times, distinct pharmacological activities. Here we describe a new protein complex containing phospholipaseA(2) (PLA(2)), protease, and a trypsin inhibitor. It is isolated from the venom of Daboia russelii by gel permeation chromatography, on a Sephadex G-75 column. This 44.6kDa complex exhibits only phospholipase A(2) activity. In the presence of 8M urea it is well resolved into protease (29.1kDa), PLA(2) (13kDa), and trypsin inhibitor (6.5kDa) peaks. The complex showed an LD(50) of 5.06mg/kg body weight in mice. It inhibited the frequency of spontaneous release of neurotransmitter in hippocampal neurons. It also caused peritoneal bleeding, and edema in the mouse foot pads. Interestingly, the complex caused degeneration of both the germ cells and the mouse Leydig cells of mouse testis. A significant reduction in both the diameter of the seminiferous tubules and height of the seminiferous epithelia were observed following intraperitoneal injection of the sub-lethal dose (3mg/kg body weight). This effect of the toxin is supported by the increase in the activities of acid and alkaline phosphatases and the nitric oxide content in the testes, and a decrease in the ATPase activity. Because of its potent organ atrophic effects on the reproductive organs, the toxin is named "Reprotoxin". This is the first report demonstrating toxicity to the reproductive system by a toxin isolated from snake venom.     

Synthesis and Mechanistic Studies of a Novel Homoisoflavanone Inhibitor of Endothelial Cell Growth

Preventing pathological ocular angiogenesis is key to treating retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. At present there is no small molecule drug on the market to target this process and hence there is a pressing need for developing novel small molecules that can replace or complement the present surgical and biologic therapies for these neovascular eye diseases. Previously, an antiangiogenic homoisoflavanone was isolated from the bulb of a medicinal orchid, Cremastra appendiculata. In this study, we present the synthesis of a novel homoisoflavanone isomer of this compound. Our compound, SH-11052, has antiproliferative activity against human umbilical vein endothelial cells, and also against more ocular disease-relevant human retinal microvascular endothelial cells (HRECs). Tube formation and cell cycle progression of HRECs were inhibited by SH-11052, but the compound did not induce apoptosis at effective concentrations. SH-11052 also decreased TNF-α induced p38 MAPK phosphorylation in these cells. Intriguingly, SH-11052 blocked TNF-α induced IκB-α degradation, and therefore decreased NF-κB nuclear translocation. It decreased the expression of NF-κB target genes and the pro-angiogenic or pro-inflammatory markers VCAM-1, CCL2, IL8, and PTGS2. In addition SH-11052 inhibited VEGF induced activation of Akt but not VEGF receptor autophosphorylation. Based on these results we propose that SH-11052 inhibits inflammation induced angiogenesis by blocking both TNF-α and VEGF mediated pathways, two major pathways involved in pathological angiogenesis. Synthesis of this novel homoisoflavanone opens the door to structure-activity relationship studies of this class of compound and further evaluation of its mechanism and potential to complement existing antiangiogenic drugs.     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

Chronic Ethanol Increases the Cannabinoid Receptor Agonist Anandamide and Its Precursor N-Arachidonoylphosphatidylethanolamine in SK - N - SH Cells

Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [³H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [³H]AnNH and [³H]N-ArPE (p < 0.05) (1.43-fold for [³H]AnNH and 1.65-fold for [³H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [³H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [³H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [³H]AnNH. The formation of [³H]AnNH and [³H]N-ArPE by EtOH was enhanced by the Ca²⁺ ionophore A23187 or by the depolarizing agent veratridine and the K⁺ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [³H]AnNH and [³H]N-ArPE was inhibited by exogenous AnNH, whereas only [³H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and G_i/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.