Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta.

The diacylglycerol lipases (DAGLα and DAGLß) hydrolyze DAG to generate 2-arachidonoylglycerol (2-AG), the principal endocannabinoid and main precursor of arachidonic acid (AA). The DAGLs make distinct tissue specific contributions toward 2-AG and AA levels, and therefore, selective modulators for these enzymes could play crucial roles toward harnessing their therapeutic potential. Relatively high-throughput assays have recently been reported for DAGLα and have proven useful toward the characterization of inhibitors of this enzyme. Similar assays are also warranted for DAGLß which was the aim of this study. We first adapted previously reported DAGLα membrane assays (using PNPB and DiFMUO as substrates) to measure recombinant DAGLß activity in membranes. In contrast to results with DAGLα, both substrates provided a relatively limited signal window for measuring DAGLß activity, however, an improved window was obtained when employing a third commercially available substrate, EnzChek. In order to further improve on the assay parameters, we successfully purified the glutathione S-transferase (GST) tagged catalytic domain of DAGLß. Activity of the enzyme was confirmed using EnzChek as well as two DAGL inhibitors (THL and OMDM-188). The purified DAGLß catalytic domain assay described here provides the basis for a relatively clean and convenient assay with the potential to be adapted for high-throughput drug discovery efforts.

A novel live cell assay to measure diacylglycerol lipase α activity.

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

Intrauterine Growth Restriction: Effects on Neural Precursor Cell Proliferation and Angiogenesis in the Foetal Subventricular Zone.

Exposure to adverse prenatal factors can result in abnormal brain development, contributing to the aetiology of several neurological disorders. Intrauterine insults could occur during neurogenesis and gliogenesis, disrupting these events. Here we investigate the effects of chronic placental insufficiency (CPI) on cell proliferation and the microenvironment in the subventricular zone (SVZ). At 30 days of gestation (DG; term ∼67 DG), CPI was induced in pregnant guinea pigs via unilateral uterine artery ligation to produce growth-restricted (GR) foetuses (n = 7); controls (n = 6) were from the unoperated horn. At 60 DG, foetal brains were stained immunohistochemically to identify proliferating cells (Ki67), immature neurons (polysialylated neuronal cell adhesion molecule), astrocytes (glial fibrillary acidic protein), microglia (ionised calcium-binding adaptor molecule-1, Iba-1) and the microvasculature (von Willebrand factor) in the SVZ. There was no overall difference (p > 0.05) in the total number of Ki67-immunoreactive (IR) cells, the percentage of SVZ occupied by blood vessels or the density of Iba-1-IR microglia in control versus GR foetuses. However, regression analysis across both groups revealed that both the number of Ki67-IR cells and the percentage of SVZ occupied by blood vessels in the ventral SVZ were negatively correlated (p < 0.05) with brain weight. Furthermore, in the SVZ (dorsal and ventral) the density of blood vessels positively correlated (p < 0.05) with the number of Ki67-IR cells. Double-labelling immunofluorescence suggested that the majority of proliferating cells were likely to be neural precursor cells. Thus, we have demonstrated an association between angiogenesis and neurogenesis in the foetal neurogenic niche and have identified a window of opportunity for the administration of trophic support to enhance a neuroregenerative response.

The antiallergic mast cell stabilizers lodoxamide and bufrolin as the first high and equipotent agonists of human and rat GPR35.

Lack of high potency agonists has restricted analysis of the G protein-coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.

The perceptions of people with multiple sclerosis about the NHS provision of physiotherapy services.

PURPOSE: In 2008, the Royal College of Physicians (RCP) and MS Trust conducted a second audit of NHS services for people with MS (PwMS). Using the free text comments obtained from the RCP and MS Trust audit, this study aimed to perform content analysis on the views of PwMS about MS services, focussing on physiotherapy provision. METHOD: A total of 757 PwMS included a free text comment on MS services and an additional 41 commented separately about physiotherapy services. The data were analysed using content analysis, which identified three main themes, positive, negative and neutral and subsequent categories and sub-categories. Intra- and inter-rater reliability were assessed and the data were manipulated to address the research aim. RESULTS: This study found that the majority of the free text comments on MS services were negative (55%). Physiotherapy provision was rated the most negative of NHS services (38%), with the primary complaints being lack of information about services and excessive waiting times for appointments. CONCLUSION: This study has revealed that NHS physiotherapy provision is not meeting the needs of PwMS.

Intrauterine growth restriction affects the maturation of myelin.

Intrauterine growth-restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in the guinea pig results in long-term deficits in brain myelination and could therefore contribute to altered neural function. CPI was induced by unilateral ligation of the uterine artery at mid-gestation (term~67 days of gestation; dg), producing growth-restricted (GR) foetuses (60 dg), neonates (1 week) and young adults (8 week); controls were from the unligated horn or sham-operated animals. In GR foetuses (n=8) and neonates (n=7), white matter (WM) volume was reduced (p<0.05); this reduction did not persist in young adults (n=11) however the corpus callosum width was reduced (p<0.05). Immunoreactivity (IR) for myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and myelin proteolipid protein (PLP), all markers of myelinating oligodendrocytes (OL), was reduced in GR foetuses compared to controls. MBP was the most markedly affected with an abnormal retention of protein in the OL soma and a reduction of its incorporation into the myelin sheath. MAG-IR OL density was reduced (p<0.05), while the density of OLs immunoreactive for Olig-2, a transcription factor expressed throughout the entire OL lineage, was increased (p<0.05). MBP-, MAG- and PLP-IR recovered to control levels postnatally. These results suggest that IUGR transiently delays OL maturation and myelination in utero but that myelination and WM volume are restored to control levels postnatally. Long-term deficits in myelination are therefore unlikely to be the major factor underlying the altered neurological function which can be associated with IUGR.

Fluoxetine reverses the memory impairment and reduction in proliferation and survival of hippocampal cells caused by methotrexate chemotherapy.

RATIONALE: Adjuvant cancer chemotherapy can cause long-lasting, cognitive deficits. It is postulated that these impairments are due to these drugs targeting neural precursors within the adult hippocampus, the loss of which has been associated with memory impairment. OBJECTIVES: The present study investigates the effects of the chemotherapy, methotrexate (MTX) on spatial working memory and the proliferation and survival of the neural precursors involved in hippocampal neurogenesis, and the possible neuroprotective properties of the antidepressant fluoxetine. METHODS: Male Lister hooded rats were administered MTX (75 mg/kg, two i.v. doses a week apart) followed by leucovorin rescue (i.p. 18 h after MTX at 6 mg/kg and at 26, 42 and 50 h at 3 mg/kg) and/or fluoxetine (10 mg/kg/day in drinking water for 40 days). Memory was tested using the novel location recognition (NLR) test. Using markers, cell proliferation (Ki67) and survival (bromodeoxyuridine/BrdU), in the dentate gyrus were quantified. RESULTS: MTX-treated rats showed a cognitive deficit in the NLR task compared with the vehicle and fluoxetine-treated groups. Cognitive ability was restored in the group receiving both MTX and fluoxetine. MTX reduced both the number of proliferating cells in the SGZ and their survival. This was prevented by the co-administration of fluoxetine, which alone increased cell numbers. CONCLUSIONS: These results demonstrate that MTX induces an impairment in spatial working memory and has a negative long-term effect on hippocampal neurogenesis, which is counteracted by the co-administration of fluoxetine. If translatable to patients, this finding has the potential to prevent the chemotherapy-induced cognitive deficits experienced by many cancer survivors.