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.

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.

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.

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.

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.

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.

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes.

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble ("ablate') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.

Analysis of the glucose transporter compliment of metabolically important tissues from the Milan hypertensive rat.

Hypertension is frequently associated with peripheral insulin resistance. An expanding body of evidence has described aberrant expression of glucose transporters in the insulin resistance associated with diabetes mellitus. Therefore, we have investigated the relative levels of expression and subcellular distribution of four members of the facilitative glucose transporter family in metabolically important tissues from the hypertensive Milan rat. Skeletal muscle is the major site of peripheral glucose disposal; skeletal muscle membranes isolated from hypertensive animals exhibited a profoundly reduced level of GLUT4 protein compared to normotensive control animals This reduction was confined to the intracellular pool which exhibited a 50% lower level of GLUT4. In contrast, adipocytes, the other major site of peripheral glucose disposal, exhibited no change in the levels of expression of either GLUT1 or GLUT4 transporter isoforms. Hepatocytes from hypertensive animals exhibit similar levels of GLUT2 protein to the normotensive controls. Patterns of expression of GLUT1, GLUT3 and GLUT4 as determined by immunoblot analysis were profoundly altered in certain brain regions in the hypertensive state. Given the importance of the GLUT4 isoform in mediating the insulin-stimulated disposal of glucose into peripheral tissues, the observation that muscle exhibits profoundly decreased levels of this transporter has important implications for the insulin-resistance associated with hypertension in these animals.

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.

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.

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.