Chronic ethanol exposure and withdrawal influence NMDA receptor subunit and splice variant mRNA expression in the rat cerebral cortex

Chronic ethanol exposure and subsequent withdrawal are known to change NMDA receptor activity. This study examined the effects of chronic ethanol administration and withdrawal on the expression of several NMDA receptor subunit and splice variant mRNAs in the rat cerebral cortex. Ethanol dependence was induced by ethanol vapour exposure. To delineate between seizure-induced changes in expression during withdrawal and those due to withdrawal per se, another group of naive rats was treated with pentylenetetrazol (PTZ) injection (30 mg/kg, i.p.). RNA samples from the cortices of chronically treated and withdrawing animals were compared to those from pairfed controls. Changes in NMDA receptor mRNA expression were determined using ribonuclease protection assays targetting the NR2A, -2B, -2C and NR1-pan subunits as well as the three alternatively spliced NR1 inserts (NR1-pan describes all the known NR1 splice variants generated from the 5' insert and the two 3' inserts). The ratio of NR1 mRNA incorporating the 5' insert vs, that lacking it was decreased during ethanol exposure and up to 48 h after withdrawal. NR2B mRNA expression was elevated during exposure, but returned to control levels 18 h after withdrawal. Levels of NR2A, NR2C, NR1-pan and both 3' NR1 insert mRNAs from the ethanol-treated groups did not alter compared with the pair-fed control group. No changes in the level of any NMDA receptor subunit mRNA was detected in the PTZ-treated animals. These data support the hypothesis that changes in NMDA receptor subunit composition may underlie a neuronal adaptation to the chronic ethanol-inhibition and may therefore be important in the precipitation of withdrawal hyperactivity. (C) 1999 Elsevier Science B.V. All rights reserved.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Free radicals in alcoholic myopathy: Indices of damage and preventive studies

Chronic alcoholic myopathy affects up to two-thirds of all alcohol misusers and is characterized by selective atrophy of Type If (glycolytic, fast-twitch, anaerobic) fibers. In contrast, the Type I fibers (oxidative, slow-twitch, aerobic) are relatively protected. Alcohol increases the concentration of cholesterol hydroperoxides and malondialdehyde-protein adducts, though protein-carbonyl concentration levels do not appear to be overtly increased and may actually decrease in some studies. In alcoholics, plasma concentrations of a-tocopherol may be reduced in myopathic patients. However, a-tocopherol supplementation has failed to prevent either the loss of skeletal muscle protein or the reductions in protein synthesis in alcohol-dosed animals. The evidence for increased oxidative stress in alcohol-exposed skeletal muscle is thus inconsistent. Further work into the role of ROS in alcoholic myopathy is clearly warranted. (C) 2002 Elsevier Science Inc.

CDT, GGT, and AST as markers of alcohol use: The WHO/ISBRA Collaborative Project

Background: Estimates of the performance of carbohydrate deficient transferrin (CDT) and gamma glutamyltransferase (GGT) as markers of alcohol consumption have varied widely. Studies have differed in design and subject characteristics. The WHO/ISBRA Collaborative Study allows assessment and comparison of CDT, GGT, and aspartate aminotransferase (AST) as markers of drinking in a large, well-characterized, multicenter sample. Methods: A total of 1863 subjects were recruited from five countries (Australia, Brazil, Canada, Finland, and Japan). Recruitment was stratified by alcohol use, age, and sex. Demographic characteristics, alcohol consumption, and presence of ICD-10 dependence were recorded using an interview schedule based on the AUDADIS, CDT was assayed using CDTect(TM) and GGT and AST by standard methods. Statistical techniques included receiver operating characteristic (ROC) analysis. Multiple regression was used to measure the impact of factors other than alcohol on test performance. Results: CDT and GGT had comparable performance on ROC analysis, with AST performing slightly less well. CDT was a slightly but significantly better marker of high-risk consumption in men. All were more effective for detection of high-risk rather than intermediate-risk drinking. CDT and GGT levels were influenced by body mass index, sex, age, and smoking status. Conclusions: CDT was little better than GGT in detecting high- or intermediate-risk alcohol consumption in this large, multicenter, predominantly community-based sample. As the two tests are relatively independent of each other, their combination is likely to provide better performance than either test alone, Test interpretation should take account sex, age. and body mass index.

Molecular introduction to head and neck cancer (HNSCC) carcinogenesis

Of all human cancers, HNSCC is the most distressing affecting pain, disfigurement, speech and the basic survival functions of breathing and swallowing. Mortality rates have not significantly changed in the last 40 years despite advances in radiotherapy and surgical treatment. Molecular markers are currently being identified that can determine prognosis preoperatively by routine tumour biopsy Leading to improved management of HNSCC patients. The approach could help decide which early stage patient should have adjuvant neck dissection and radiotherapy, and whether Later stage patients with operable lesions would benefit from resection and reconstructive surgery or adopt a conservative approach to patients with poor prognosis regardless of treatment. In the future, understanding these basic genetic changes in HNSCC would be important for the management of HNSCC. (C) 2004 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.

Alternative polyadenylation and splicing of mRNAs transcribed from the human Sin1 gene

Limited but significant sequence similarity has been observed between an uncharacterized human protein, SIN1, and the S. pombe SIN1, Dictyostelium RIP3 and S. cerevisiae AVO1 proteins. The human Sin1 gene has been automatically predicted (MAPKAP1; GenBank accession number NM_024117); however, this sequence appears to be incomplete. In this study, we have cloned and characterized the full-length human Sin1 mRNA and identified a highly conserved domain that defines the family of SIN1 orthologues, members of which are widely distributed in the fungal and metazoan kingdoms. We demonstrate that Sin1 transcripts can use alternative polyadenylation signals and describe a number of Sin1 splice variants that potentially encode functionally different isoforms. (C) 2004 Elsevier B.V. All rights reserved.

Promoter-, cell-, and ligand-specific transactivation responses of the VDRB1 isoform

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D-3, the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D-3 was greater in Cost and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D-3-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D-3 and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D-3 than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand. (c) 2005 Elsevier Inc. All rights reserved.

Neuronal voltage-gated sodium channel subtypes: Key roles in inflammatory and neuropathic pain

Voltage-gated sodium channels (VGSCs) play an important role in neuronal excitability. Regulation of VGSC activity is a complex phenomenon that occurs at multiple levels in the cell, including transcriptional regulation, post-translational modification and membrane insertion and retrieval. Multiple VGSC subtypes exist that vary in their biophysical and pharmacological properties and tissue distribution. Any alteration of the VGSC subtype profile of a neuron or the mechanisms that regulate VGSC activity can cause significant changes in neuronal excitability. Inflammatory and neuropathic pain states are characterised by alterations in VGSC subtype composition and activity in sensory neurons. This review focuses on the VGSC subtypes involved in such pain states. (c) 2006 Elsevier Ltd. All rights reserved.

Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.