Moving on from responsible gambling: a new discourse is needed to prevent and minimise harm from gambling.

OBJECTIVES: To address the current status of responsible gambling (RG) as the dominant discourse for reducing gambling harm. STUDY DESIGN: The article is a narrative review of relevant literature analysed discursively. METHODS: We identified significant texts describing the discourse of RG and analysed these to extract major characteristics and themes of the discourse. These were then subjected to a critique, using the public health discourses as an alternative system for addressing gambling harm. RESULTS: The discourse of RG is inadequate for preventing or minimising gambling harm. A public health-focused approach to prevent and minimise gambling harm is likely to be far more effective but will be opposed by vested interests. CONCLUSIONS: It is timely to consider abandoning the discourse of RG. This discourse has been discredited because of its complicity with vested interests and a lack of evidence to demonstrate its efficacy in preventing or reducing harm. A public health response to the prevention of gambling harm is feasible and practical and can and should be further developed and implemented rapidly.

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle-associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.

Altered G-protein expression and adenylate cyclase activity in platelets of non-insulin-dependent diabetic (NIDDM) male subjects.

Adenylate cyclase activity and levels of guanine nucleotide regulatory proteins (G-proteins) were compared in platelets from normal and non-insulin-dependent diabetic (NIDDM) male subjects. Whilst no differences were noted in basal and NaF-stimulated adenylate cyclase activities the degree of stimulation achieved by both forskolin and prostaglandin, E1 was lower by some 34 and 52% respectively, in platelet membranes from diabetic subjects compared with those from normal control subjects. Altered alpha 1-adrenoceptor-mediated inhibition of prostaglandin E1-stimulated adenylate cyclase activity was evident; it being some 34% lower in platelet membranes from diabetic subjects compared to controls. Analysis of G-protein alpha-subunits, using specific anti-peptide antisera, showed that platelets from all subjects exhibited the Gi-2 and Gi-3, but not the Gi-1 forms of the inhibitory G-protein 'Gi' and all expressed the 42 kDa species of alpha-subunit of the stimulatory G-protein Gs. Whilst platelets of diabetic subjects had levels of Gs which were comparable to those found in control subjects their levels of Gi-2 and Gi-3 were some 49 and 75%, respectively, of those found in platelets from control subjects. It is suggested that changes in adenylate cyclase functioning and G-protein expression may contribute to altered platelet functioning in non-insulin-dependent diabetic subjects.

Heat shock protein antibody titers are reduced by statin therapy in dyslipidemic subjects: a pilot study.

Antibody titers to heat shock protein (Hsp)-60 and -65 are positively related to risk of vascular disease and cardiovascular endpoints. There are few data on the factors that regulate the levels of these antibodies. It is known that the statins have antiinflammatory and immunoregulatory properties. The authors examined the effects of 2 statins, simvastatin (Zocor) and atorvastatin (Lipitor) on antibody titers to Hsp-60, -65, and -70 in a group of dyslipidemic patients. Twenty patients attending a lipid clinic, and previously not receiving lipid-lowering treatment, were treated with 10 mg of simvastatin (n = 11) or atorvastatin (n = 9) for 4 months. An additional 14 patients were recruited from the same clinic at the same hospital as a control group. The medication of these latter patients was unaltered for 4 months and the same parameters were measured as for the statin group. Antibody titers to Hsp-60, -65, and -70 were measured by enzyme-linked immunosorbent assay and lipoprotein profile and highly sensitive serum C-reactive protein (CRP) were measured by routine methods before and after treatment. Pretreatment and posttreatment data were compared by paired t or Mann-Whitney tests. Overall statin treatment was associated with a significant reduction in median antibody titers to Hsp-60 (17.2%, p = 0.03), Hsp-65 (15.9%, p = 0.003) and Hsp-70 (8.3%, p = 0.006), but not in control patients. Both statins caused a reduction in median serum CRP concentrations (45% overall, p < 0.05), but significant changes were not observed in the control patients. The effects on Hsp antibody titers were not related to changes in serum CRP concentrations (p > 0.05). However, there was a significant correlation between changes in antibody titers to Hsp-60 vs Hsp-65 (p < 0.01), Hsp-60 vs Hsp-70 (p < 0.05), and Hsp-65 vs Hsp-70 (p < 0.001). Statin treatment was associated with a reduction in antibody titers to Hsp-60, -65, and -70. This reduction is not fully explained by the antiinflammatory effects of the statins but may be due to their other immunomodulatory properties.

Dynamics of insulin-stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein.

Insulin increases glucose uptake by promoting the translocation of the GLUT4 isoform of glucose transporters to the plasma membrane. We have studied this process in living single cells by fusing green fluorescent protein (GFP) to the N-terminus (GFP-GLUT4) or C-terminus (GLUT4-GFP), of GLUT4. Both chimeras were expressed in a perinuclear compartment of CHO cells, and in a vesicular distribution through the cytosol. Insulin promoted an increase in plasma membrane fluorescence as a result of net translocation of the chimeras to the cell surface. GLUT4-GFP, but not GFP-GLUT4, was re-internalised upon the removal of insulin suggesting that a critical internalisation signal sequence exists in the N-terminus of GLUT4. The use of GFP thus allows an analysis of GLUT4 trafficking in single living cells.

Hypothalamic GLUT 4 expression: a glucose- and insulin-sensing mechanism?

The insulin-regulatable glucose transporter, GLUT 4, is expressed primarily in peripheral tissues (skeletal muscle and adipose tissue). In response to insulin this transporter moves rapidly from an intracellular storage site to the plasma membrane, thus accounting for the substantial increase in glucose uptake by these tissues following insulin stimulation. The recent finding that GLUT 4 is also expressed in the hypothalamus suggests that this brain region, which is outside the blood-brain barrier and therefore sensitive to circulating insulin, may experience stimulation of glucose uptake in response to insulin. We propose that this may allow regions of the hypothalamus to respond directly to elevated blood glucose, constituting a form of metabolic regulation by allowing circulating glucose (and therefore insulin) in concert with other mechanisms to maintain blood glucose homeostasis. We consider the possible physiological role of such a mechanism and speculate that disturbances of this mechanism may occur in endocrine disease associated with insulin resistance.

The ischaemic lactate-ammonia test.

The ischaemic lactate-ammonia test is widely used for investigating patients with muscle pain and fatigue. It involves measuring plasma lactate and ammonia produced as a result of forearm exercise under ischaemic conditions in a fasted subject. Its clinical use is to screen patients with muscle complaints for disorders of carbohydrate metabolism, in particular to identify those in whom further investigation may provide useful diagnostic information. There is a wide variety of methods described, reflecting attempts to optimize the response and hence the diagnostic value of the test. Although it is often considered a general screening test for metabolic muscle disease, the situations in which it is useful are specific. Here we review the use of the test and present the results of an audit of its use in our departments.

Radioimmunoassays of arginine vasopressin and atrial natriuretic peptide: application of a common protocol for plasma extraction using Sep-Pak C18 cartridges.

A rapid vacuum-driven procedure, using pre-treated Sep-Pak C18 cartridges, has been developed for the simultaneous extraction of arginine vasopressin (AVP) and atrial natriuretic peptide (ANP) from plasma. Non-specific interference was removed by fractional elution with an aqueous methanol/trifluoroacetic acid (TFA) mixture. AVP and ANP were coeluted under positive pressure with a methanol/TFA mixture and the eluates air-dried before measurement using separate radioimmunoassays. Assay ranges for AVP and ANP were 0.12-29.5 pmol/L and 0.65-162 pmol/L, respectively, with mean recoveries (standard deviation in parentheses) for AVP of 96.4% (5.5%) at a level of 11.8 pmol/L and for ANP of 94.8% (5.9%) at a level of 32.4 pmol/L. The extraction and assay procedures were validated by observing the changes in plasma AVP and ANP concentrations in normal subjects at different stages of hydration and in elderly patients during treatment for congestive cardiac failure.

Model systems for the evaluation of mucolytic drugs: acetylcysteine and S-carboxymethylcysteine.

The therapeutic place of mucolytic drugs remains uncertain; clinical studies have seldom demonstrated significant benefit and the activity of such agents is poorly understood. In this study the effects of the mucolytic agents acetylcysteine (AC) and S-carboxymethylcysteine (SCMC) have been assessed in-vitro, using purified mucus gels and tracheal explant systems and in-vivo, in the mini-pig tracheal pouch model, in order to elucidate their mechanisms of action. A reduction in the elastic modulus (up to 70% over the frequency range 0.2-20 Hz) was apparent after treatment of mucus gels in-vitro with AC (P less than 0.05), but not with SCMC. Gel chromatography indicated that AC reduced the mucus glycoprotein to smaller subunits and a breakdown of gel structure was apparent when visualized using a cryofracture technique. SCMC treated gels were comparable with control samples. Mucus production was assessed in isolated rat trachea by monitoring the uptake and release of [3H] glucosamine. AC (5-15 mM) did not affect secretion whereas SCMC (5 and 10 mM) reduced the production of radiolabelled material (24 and 37%, respectively) over 24 h (P less than 0.05). Single oral doses of SCMC and AC (20 mg kg-1) were administered to mini-pigs and mucus collected from tracheal pouches; no significant changes in the rheological or biochemical properties of the secretion could be determined. The in-vitro mucolytic activity of AC depends upon a direct action on the secretion, SCMC appears able to reduce production of the mucus glycoprotein. Wide inter- and intra-individual variation in the properties of the secretion would suggest that such effects are not readily demonstrated in-vivo.

Plasma antibody titres to heat shock proteins-60, -65 and-70: their relationship to coronary risk factors in dyslipidaemic patients and healthy individuals.

OBJECTIVE: To investigate the factors that may affect antibody titres to heat shock proteins (Hsp)-60, -65 and -70, and serum C-reactive protein (CRP) concentrations in patients with dyslipidaemia and other features of the metabolic syndrome as defined by ATPIII criteria. MATERIAL AND METHODS: The study comprised 237 dyslipidaemia patients and 135 healthy individuals recruited from amongst university and hospital employees. RESULTS: Compared to the healthy individuals, the dyslipidaemic patients had higher antibody titres to Hsp-60 (p<0.01), Hsp-65 (p<0.001) and Hsp-70 (p<0.05), and higher serum CRP concentrations (p<0.001). The best-fitting multifactorial models revealed that known coronary risk factors explained little of the variation in Hsp antibody titres: 3 % for Hsp-60, 1 % for Hsp-65 and 4 % for Hsp-70 amongst the dyslipidaemic subjects. The corresponding values for the subgroup with the metabolic syndrome were 8 %, 3 % and 1 %, respectively. In contrast, the best-fitting model explained 13.5 % of the variation in serum CRP concentrations among the dyslipidaemic patients, obesity being a major determinant; and 14 % in the subgroup with metabolic syndrome. CONCLUSIONS: The higher antibody titres to Hsp-60, -65, and -70 in the dyslipidaemic patients may be related to a heightened state of immunoactivation associated with atherosclerosis in this group. Our data indicate that antibody titres to these Hsps are not associated with the classical coronary risk factors, although serum high sensitivity (hs)CRP concentrations were significantly related to obesity.

Secondary structure prediction from multiple sequence data: blood clotting factor XIII and Yersinia protein-tyrosine phosphatase.

Predictions of protein structure are best tested without prior knowledge of the protein three-dimensional structure. Three-dimensional atomic models will soon be determined by X-ray crystallography for the alpha-subunit of human blood clotting factor XIII and members of the family of protein tyrosine specific phosphatases. Accordingly, we here present secondary structure predictions for each of these proteins. The secondary structure predictions were generated from aligned sets of protein sequences. This technique has previously provided reliable predictions for the Annexins and the SH2 domains. The factor XIII alpha prediction contains 39 regions predicted in strand conformation (34% of the protein) with only 3 helices (4%). The protein tyrosine phosphatases have 12 predicted strands and 5 helices (30 and 17%, respectively). We expect greater reliability from regions of alignments that show clear patterns of residue conservation (61% of factor XIII alpha and 57% of the protein tyrosine phosphatases). The aligned protein tyrosine phosphatases show two regions (L39-L80 and I138-E253) with clear patterns of residue conservation separated by a region of variable amino acid composition. We suggest this indicates that the tyrosine phosphatase fold comprises two domains separated by an exposed linker. Potential phosphate binding sites are identified in the protein tyrosine phosphatases.

Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation.

An algorithm is described for the systematic characterization of the physico-chemical properties seen at each position in a multiple protein sequence alignment. The new algorithm allows questions important in the design of mutagenesis experiments to be quickly answered since positions in the alignment that show unusual or interesting residue substitution patterns may be rapidly identified. The strategy is based on a flexible set-based description of amino acid properties, which is used to define the conservation between any group of amino acids. Sequences in the alignment are gathered into subgroups on the basis of sequence similarity, functional, evolutionary or other criteria. All pairs of subgroups are then compared to highlight positions that confer the unique features of each subgroup. The algorithm is encoded in the computer program AMAS (Analysis of Multiply Aligned Sequences) which provides a textual summary of the analysis and an annotated (boxed, shaded and/or coloured) multiple sequence alignment. The algorithm is illustrated by application to an alignment of 67 SH2 domains where patterns of conserved hydrophobic residues that constitute the protein core are highlighted. The analysis of charge conservation across annexin domains identifies the locations at which conserved charges change sign. The algorithm simplifies the analysis of multiple sequence data by condensing the mass of information present, and thus allows the rapid identification of substitutions of structural and functional importance.

ATF-2 contains a phosphorylation-dependent transcriptional activation domain.

The ATF-2 transcription factor can mediate adenovirus E1A-inducible transcriptional activation. Deletion analysis has indicated that the N-terminal region of ATF-2 is essential for this response. Furthermore, the N-terminus can activate transcription in the absence of E1A when fused to a heterologous DNA binding domain. However, in the intact protein this activation domain is masked. In this report we show that residues in the N-terminus required for activation are also required for mediating E1A stimulation. In particular two threonine residues at positions 69 and 71 are essential. These residues are phosphorylated in vivo and can be efficiently phosphorylated in vitro by the JNK/SAPK subgroup of the MAPK family. ATF-2 can bind to a UV-inducible kinase through a region in the N-terminus that is distinct from the sites of phosphorylation; this binding region is both necessary for phosphorylation by JNK/SAPK in vitro and for transcriptional activation in vivo. The activity of the N-terminus is stimulated by UV irradiation which stimulates the signalling pathway leading to JNK/SAPK. Finally, although ATF-2 binds to the E1A protein, the N-terminal activation domain is not required for this interaction. The results show that ATF-2, like other members of the ATF/CREB family of DNA binding proteins is regulated by specific signalling pathways.

Lipid dynamics and lipid-protein interactions in rat hepatocyte plasma membranes.

Rat hepatocyte plasma membranes were isolated and examined by differential scanning calorimetry and by steady state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, 12-anthroyl stearate, and 2-anthroyl stearate. Calorimetry of the intact membranes revealed a reversible lipid thermotropic transition with lower and upper critical temperatures of 18 degrees and 31 degrees C, respectively. The transition was also observed in lipid extracts of the membrane, both dried and rehydrated. The transition enthalpies estimated for intact membranes, dried membrane lipids, and rehydrated lipids, respectively, were approximately 0.3, 1.2 to 1.4, and 0.5 to 0.7 cal/g of lipid. The transition observed in the native membranes is broad and of low enthalpy, owing in part to the relatively high cholesterol content and to protein-lipid interactions. Fluorescence polarization studies detect the lower critical temperature of the transition in the membranes and in sonicated dispersions of the membrane lipid. Arrhenius studies of membrane 5'-nucleotidase and alkaline phosphatase give two-slope plots with breakpoints, respectively, of approximately 17 degrees and 26 degrees C. These break points and others reported for a number of hepatocyte membrane protein activities cluster uniformly at either the lower or the upper critical temperature of the lipid transition observed by differential scanning calorimetry.

Calcium modulates the lipid dynamics of rat hepatocyte plasma membranes by direct and indirect mechanisms.

Calcium ion decreases the motional freedom of lipid molecules in isolated rat hepatocyte plasma membranes and in sonicated dispersions (liposomes) of the membrane lipid. The decrease in lipid fluidity was monitored by estimation of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. At least two processes are involved in the mode of action of the cation. The first is direct, i.e., observed on addition of calcium to the liposomes, relatively rapid, with a half-time of 10-15 at 37 degrees C, proportional to the calcium concentration in the range 0-4 mM, and readily reversed on addition of excess EDTA. The second mechanism is indirected and requires the presence of the membrane proteins. It occurs relatively slowly, with a half-time of 75 min at 37 degrees C, tends to plateau with a calcium half-saturation concentration of approximately 1 mM, is of greater magnitude than the direct effect, and cannot be reversed on chelation of calcium by EDTA. Moreover, the indirect effect is specific for Ca2+ as compared to other divalent cations and it results in changes in the lipid composition. Stimulation of phospholipase A activity is likely but does not account for the change in fluidity. The direct action of calcium is ascribed to binding to the lipid bilayer, whereas the indirect action probably results from modulation of membrane-bound enzymes which can alter the lipid composition. The effects of calcium on the membrane lipid fluidity may underly certain of its regulatory actions on membrane functions.