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Emphysematous or gas-forming infections, a very small percentage of bacterial infections of the urinary tract, attract importance because of their life threatening potential. Herein, we report a 60-year-old Saudi female patient who was a known case of Diabetes mellitus for 15 years. She was admitted with left flank pain of 5 days duration, abdominal distension, nausea, vomiting and chills associated with increased frequency of urine, urgency, and dysuria. She had leukocytosis, high blood sugar, elevated urea and creatinine and pyuria. Urine culture grew Escherichia coli. Ultrasound and CT scan showed left pelvicalyceal dilatation and air in the left kidney and urinary bladder. She was treated with a prolonged parenteral antibiotic course, and insulin, with complete recovery.

Genome-wide experimental methods to identify disease genes, such as linkage analysis and association studies, generate increasingly large candidate gene sets for which comprehensive empirical analysis is impractical. Computational methods employ data from a variety of sources to identify the most likely candidate disease genes from these gene sets. Here, we review seven independent computational disease gene prioritization methods, and then apply them in concert to the analysis of 9556 positional candidate genes for type 2 diabetes (T2D) and the related trait obesity. We generate and analyse a list of nine primary candidate genes for T2D genes and five for obesity. Two genes, LPL and BCKDHA, are common to these two sets. We also present a set of secondary candidates for T2D (94 genes) and for obesity (116 genes) with 58 genes in common to both diseases.

G protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensitized. One of the key events in receptor desensitization is the sequestration of the receptor from the cell surface into acidic intracellular endosomes. Therefore, a convenient, generic, and noninvasive monitor of this process is desirable. A novel, pH-sensitive, red-excited fluorescent dye, CypHer 5, was synthesized. This dye is non-fluorescent at neutral pH and is fluorescent at acidic pH. Anti-epitope antibodies labeled with this dye were internalized in an agonist concentration- and time-dependent manner, following binding on live cells to a range of GPCRs that had been modified to incorporate the epitope tags in their extracellular N-terminal domain. This resulted in a large signal increase over background. When protonated, the red fluorescence of CypHer 5 provides a generic reagent suitable for monitoring the internalization of GPCRs into acidic vesicles. This approach should be amenable to the study of many other classes of cell surface receptors that also internalize following stimulation.

Mouse neuroblastoma x rat glioma hybrid NG 108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human beta 2 adrenoceptor cDNA under the control of the beta actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (beta N22) but not one expressing only low levels of the receptor (beta N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg2+ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn2+. The elevated basal activity was partially reversed by addition of the beta-adrenoceptor antagonist propranolol. Agonist activation of beta N22 but not beta N17 cells led to a large selective down-regulation of cellular Gs alpha levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [3H] forskolin was achieved with substantially greater potency (some 30 fold) in beta N22 cell membranes than in beta N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [3H] forskolin binding which was equipotent in each of beta N22, beta N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of Gs alpha down-regulation in each cell type. Expression of an epitope tagged variant of Gs alpha in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type Gs alpha. Isolation of clones of NCB20 cells expressing high levels of the beta 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of Gs alpha.

Clones of neuroblastoma x glioma hybrid, NH108-15, cells expressing differing levels of the human beta 2 adrenoceptor were isolated. Two clones were examined in detail, beta N22 which expressed some 4000 fmol/mg of membrane protein and clone beta N17 which expressed approx. 300 fmol/mg of membrane protein of the receptor. In beta N22 cells 'basal' adenylate cyclase activity measured in the presence of Mg2+ was significantly greater than that in wild-type NG108-15 or beta N17 cells. Both isoprenaline and iloprost were able to stimulate adenylate cyclase activity in each of beta N22 and beta N17 membranes. However, the EC50 for isoprenaline stimulation of adenylate cyclase in membranes of beta N22 cells (6 nM) was significantly lower than that in membranes of beta N17 cells (80 nM), whereas the EC50 for iloprost stimulation of adenylate cyclase (approx. 25 nM) was the same in the two clones and in parental NG108-15 cells. The high basal adenylate cyclase activity of beta N22 cell membranes was not a reflection of higher levels of expression of the adenylate cyclase catalytic unit, as adenylate cyclase activity measured in the presence of Mn2+ was equivalent in membranes of each of wild-type NG108-15 cells and clones beta N22 and beta N17. Basal adenylate cyclase activity measured in the presence of Mg2+ in clone beta N22 was significantly reduced, however, by the beta-receptor antagonist propranolol, whereas this agent was without effect on basal adenylate cyclase activity in membranes of wild-type NG108-15 cells. These data indicate that the elevated basal adenylate cyclase cascade in NG108-15 cells expressing high levels of the beta 2 adrenoceptor represents empty receptor activation of the signalling cascade.

Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, Gs. Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of Gs alpha. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human beta 2-adrenoceptor cDNA under the control of the beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of beta N22 cells to the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-protein alpha subunits, including Gs alpha. In beta N22 cells the IP prostanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down-regulation of cellular Gs alpha levels. Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.

To understand the details of regulation of guanine-nucleotide-binding-protein-linked transmembrane cellular-signalling cascades, it is important to know the absolute levels of each polypeptide component and the stoichiometry of their interactions. Amounts of the IP prostanoid receptor, the stimulatory G protein of the adenylyl cyclase cascade (Gs alpha) and the functional complex of Gs alpha with adenylyl cyclase, which acts as the cyclic AMP generator, were measured in membranes of neuroblastoma x glioma hybrid, NG108-15, cells. As measured by the specific binding of [3H]prostaglandin E1, the IP prostanoid receptor was present in some 100,000 copies/cell. Gs alpha assessed by quantitative immunoblotting with recombinantly expressed protein, was present in considerably higher levels (1,250,000 copies/cell). However, the maximal formation of a complex of Gs alpha and adenylyl cyclase represented only some 17,500 copies/cell. The previously established 8:1 stoichiometry of concurrent downregulation of Gs alpha and the IP prostanoid receptor in these cells [Adie, E. J., Mullaney, I., McKenzie, F. R. & Milligan, G. (1992) Biochem. J. 285, 529-536] indicates that full-agonist occupation of the receptor should be able to activate some 65% of the expressed Gs. Despite the potential 70-fold excess of Gs alpha over the Gs alpha/adenylyl cyclase complex, IP prostanoid-receptor-agonist-mediated reduction of Gs alpha levels by some 35% resulted in a 25% reduction in the maximal formation of the Gs alpha/adenylyl cyclase complex. Such results demonstrate that adenylyl cyclase is quantitatively the least highly expressed component of this signalling cascade and suggests that much of the cellular Gs alpha may not have access to adenylyl cyclase.

Neuroblastoma x glioma hybrid NG108-15 cells express a high-affinity IP prostanoid receptor. Saturation binding analysis of this receptor, using [3H]prostaglandin E1 ([3H]PGE1) as ligand, indicated that it was present at some 1.5 pmol/mg of membrane protein and displayed a dissociation constant for this ligand of 30-40 nM. Prolonged exposure of these cells either to PGE1 or to iloprost, which is a stable analogue of prostacyclin, caused a 40-70% decrease in levels of the receptor. The remaining receptors were capable of interacting with the stimulatory G-protein (Gs) of the adenylate cyclase cascade, as saturation analysis of the binding of [3H]PGE1 indicated that they had a similar affinity for the 3H-labelled ligand, and because the specific binding of [3H]PGE1 to these receptors was still sensitive to the presence of poorly hydrolysed analogues of GTP. We have recently demonstrated that prolonged exposure of NG108-15 ells to PGE1 causes a cyclic AMP-independent loss of Gs alpha-subunit (Gs alpha) from these cells [McKenzie & Milligan (1990) J. Biol. Chem. 265, 17084-17093]. Steady-state concentration of the larger 45 kDa form of Gs alpha (which is the predominant form expressed in these cells) was assessed to be 9.6 pmol/mg of membrane protein, and treatment with iloprost decreased levels of this polypeptide to some 3.0 pmol/mg of protein. Time courses of iloprost-mediated down-regulation of the IP prostanoid receptor, loss of Gs alpha protein as assessed by immunoblotting and loss of Gs alpha activity as assessed by the reconstitution of NaF stimulation of adenylate cyclase activity to membranes of S49 cyc- cells by sodium cholate extracts of NG108-15 cells were identical, suggesting that the loss of the IP prostanoid receptor and G-protein occurred in parallel. Each of these effects was half-maximal between 2 and 3 h of exposure to the agonist. Stoichiometry of loss of Gs alpha and IP prostanoid receptor was unchanged by the percentage receptor occupancy, and quantification indicated the loss of some 7-10 mol of Gs alpha/mol of receptor. This is the first report to demonstrate the temporal concurrence of loss of Gs alpha and of a receptor which interacts with this G-protein. Chronic activation of the IP prostanoid receptor on these cells results in the development of a heterologous form of desensitization to agents which function to activate adenylate cyclase [Kelly, Keen, Nobbs & MacDermot (1990) Br. J. Pharmacol. 99, 306-316]. Agonist regulation of Gs alpha levels in these cells may contribute to this process.