Identification and characterization of a glucagon receptor from the goldfish Carassius auratus: implications for the evolution of the ligand specificity of glucagon receptors in vertebrates

The structural basis of ligand selectivity of G protein-coupled receptors for metabolic hormones has been an area of intense investigation, and yet it remains unresolved. One approach to delineating the mechanism of ligand-receptor interactions is to compare the ligand specificities of receptors expressed in species that emerged at different times within vertebrate evolution. In this paper we describe the isolation, functional, and phylogenetic characterization of the glucagon receptor from the goldfish Carassius auratus (Teleostei, order Cypriniformes), and compare its ligand specificity with that of the homologous rat receptor. Goldfish (gf) glucagon stimulated glucose production in a dose-dependent manner from isolated goldfish hepatocytes, resulting in 5-fold increase at 1 microm. The goldfish glucagon receptor (gfGlucR) shares 56, 51, 50, and 52% amino acid identities with frog Rana tigrina regulosa, mouse, rat, and human glucagon receptors, respectively. In competitive binding experiments, the recombinant gfGlucR displays high affinity toward goldfish, zebrafish, and human glucagons (IC(50) = 0.6, 9, and 13 nm, respectively) but not toward goldfish glucagon-like peptide-1 or human glucagon-like peptide-1 (7-36) amide. Whereas both goldfish and human glucagons stimulated dose-dependent increases in intracellular cAMP through the recombinant gfGlucR, the recombinant rat GlucR interacted only with human glucagon, analogous to the specificity of the previously characterized glucagon receptor from the frog R. tigrina regulosa. Our results demonstrate that the binding pocket of gfGlucR can accommodate a broad range of glucagon structures and that in the frogs and mammals, there is a structural switch to a more restrictive conformation of glucagon receptors.     

BLOOD BORNE VIRUS—INNOVATIVE APPROACH TO EVALUATE,PREVENT CROSS‐TRANSMISSION IN DIALYSIS UNITS

Large public health interventions to control infectious disease outbreaks are common, but rigorous evaluation to improve the quality and effectiveness of these is rarely undertaken. This project aimed to prevent blood borne virus (BBV) cross‐transmission in dialysis units. Following an incident concerning the diagnosis of acute Hepatitis B in a haemodialysis patient, possibly from healthcare associated acquisition, a multifaceted and multidisciplinary investigation was conducted involving consumers, health professionals and administrations. The results of this investigation were then used to produce practical operational guidelines for planning and future interventions. To date, there has been no incidence reported of any cross‐transmission of BBV amongst our dialysis population. The actions implemented can be utilised by other departments in preventing other bacterial or viral outbreaks.     

Water and electrolyte contents, cell pH and membrane potentials of cultured turtle thyroid cells

Water and electrolyte contents, cell pH, membrane potential and 125I- uptake were determined in cultured follicular cells of turtle thyroid. The Na+, K+ and Cl- concentrations in the cultured thyroid cells were 59.2, 119.0 and 50.9 mmol/l cell water respectively. Treatment with TSH (10 mu./ml for 24 h) increased the K+ and Cl- and decreased the Na+ concentrations in cells. The water and protein contents of these cells were 81.6 and 8.7 g/100 g cells respectively. The cell pH was 6.91. With glass micro-electrodes, the resting membrane potential of thyroid cells cultured in Medium 199 averaged 33.9 +/- 0.63 mV which is slightly higher than 29.8 +/- 1.6 mV as calculated from the data on the uptakes of [14C]methyltriphenylphosphonium and 3H2O by the cells. The potential varied linearly with the log of external K+ concentration (between 15 and 120 mmol/l) with a slope of about 24 mV per tenfold change in K+ concentration. Both TSH and cyclic AMP depolarized the cell membrane. Calculations based on the values for the electrolyte concentrations in cells and in culture medium indicated that Na+, K+ and Cl- were not distributed according to their electrochemical gradients across the cell membrane. Na+ was actively transported out of the cells and K+ and Cl- into the cells. Follicular cells of turtle thyroid cultured in the medium without addition of TSH formed a monolayer. Their iodide-concentrating ability was low and they did not respond to TSH with an increase in iodide uptake. In contrast, cells cultured in medium containing TSH tended to aggregate and organize to form follicles. They had higher ability to concentrate iodide and respond to TSH.     

Localization of the isthmus in reentrant circuits by analysis of electrograms derived from clinical noncontact mapping during sinus rhythm and ventricular tachycardia

INTRODUCTION: New methods for electrogram analysis accurately estimated reentrant circuit isthmus location and shape in a canine model. It was hypothesized that these methods also would locate reentrant circuits causing clinical ventricular tachycardia (VT). METHODS AND RESULTS: Intracardiac electrogram recordings, obtained with a noncontact mapping system, were analyzed retrospectively from 14 patients with reentrant VT who had undergone successful radiofrequency ablation for prevention of VT initiation. Unipolar electrograms from 256 uniformly distributed endocardial sites were reconstructed by mathematical transformation. Twenty-seven tachycardias were mapped; 15 (in 11 patients) had a complete endocardial reentrant circuit with a figure-of-eight conduction pattern. During sinus rhythm, the location and axis of the slowest and most uniform conduction in the region of latest endocardial activation (the primary axis), the limits of which were defined as boundaries with >15 ms difference in electrogram duration between contiguous recordings, identified the location and shape of the reentrant circuit isthmus with a mean sensitivity compared with activation mapping of 79.3% and a mean specificity of 97.6%. The midpoint of a theoretical "estimated best ablation line" drawn perpendicular to the primary axis of activation, spanning the estimated isthmus location was within 1.3 +/- 0.2 cm (mean distance +/- SD) of the actual ablation site that terminated tachycardia. Analysis of VT electrograms, based on time shifts in the far-field component of the local electrogram when cycle length changed (piecewise linear adaptive template matching [PLATM] method) in 5 of the cases, accurately estimated the time interval between activation at the recording site and the circuit isthmus slow conduction zone where the effective ablation lesion had been placed, which is proportional to the distance between the two locations (mean difference compared with activation mapping: +/-37.3 ms). CONCLUSION: In selected patients with VT who have a complete endocardial circuit, isthmus location and shape can be discerned by analysis of sinus rhythm or tachycardia electrograms, and an effective ablation site can be predicted without the need to construct activation maps of reentrant circuits.     

2[125I]Iodomelatonin binding sites in guinea pig platelets

Using 2[125I]iodomelatonin as the radioligand, we characterized 2[125I]iodomelatonin binding sites in guinea pig platelet membrane preparations. Saturation radioreceptor studies indicated that these 2[125I]iodomelatonin binding sites were of picomolar affinity and femtomolar density. The dissociation constant (Kd) and maximum number of receptor sites (Bmax) were 42.5 +/- 1.79 pM and 11.8 +/- 0.8 fmol/mg protein (n = 6), respectively. 2[125I]Iodomelatonin competition studies with indoles or drugs indicate the following rank order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-methoxytryptophol, whereas serotonin and its analogs had less than 20% inhibition at 0.1 mM. Guanosine 5'-O-(3-thiotriphosphate) significantly increased the Kd by twofold suggesting that these binding sites are coupled to the guanine nucleotide binding proteins. Immunoblotting studies using anti-MT(1) IgG demonstrated one peptide blockable band with an apparent molecular mass of 37 kDa. Melatonin had no effect on prostacyclin or forskolin-stimulated intracellular 3',5'-cyclic adenosine monophosphate accumulation. A diurnal variation in binding density, which was abolished after the animals were adapted to constant light conditions, was observed. Age related studies demonstrated that Bmax increased as the animal matured. Physiological melatonin concentrations potentiated whereas those at pharmacological levels inhibited adenosine diphosphate- or arachidonic acid-stimulated platelet aggregation. Our study demonstrated G-protein coupled, saturable, reversible and highly specific picomolar affinity 2[125I]iodomelatonin binding sites in guinea pig platelets. Pharmocological and physiological data indicate that they may be different from the nanomolar [3H]melatonin binding sites in human platelets previously reported.     

Combination of retinoic acid and tumor necrosis factor overcomes the maturation block in a variety of retinoic acid-resistant acute promyelocytic leukemia cells

Retinoic acid (RA) overcomes the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. Patients receiving RA alone invariably develop RA resistance. RA-resistant cells can serve as useful models for the development of treatments for both APL and other leukemias. Previously, we showed that RA and tumor necrosis factor (TNF) promote monocytic differentiation of the APL cell line NB4 and U937 monoblastic cells. Here, we report that combining TNF with RA leads to maturation of several RA-resistant APL cells along a monocytic pathway, whereas UF-1, a patient-derived RA-resistant cell line, showed characteristics of granulocytic differentiation. We found distinct differences in gene regulation between UF-1 cells and cells showing monocytic differentiation. Although IRF-7 was up-regulated by TNF and RA in all cells tested, expression of c-jun and PU.1 correlated with monocytic differentiation. Furthermore, synergistic induction of PU.1 DNA binding and macrophage colony-stimulating factor receptor (m-CSF-1R) mRNA was observed only in cells differentiating into monocytes. Using neutralizing antibodies against m-CSF-1R or its ligand, we found that inhibiting this pathway strongly reduced CD14 expression in response to RA and TNF, suggesting that this pathway is essential for their synergy in RA-resistant leukemia cells.