Characterisation of human recombinant somatostatin receptors. 3. Modulation of adenylate cyclase activity

The five human somatostatin receptor subtypes (hsst1-5) were stably expressed in CCL39 cells (Chinese hamster lung fibroblast cells) to study the inhibition of forskolin-stimulated adenylate cyclase (FSAC) activity induced by somatostatin (somatotropin release inhibiting factor, SRIF), cortistatin (CST) and SRIF peptide analogues. Inhibition of FSAC was observed with all five receptors, although the maximal effects produced by SRIF14 varied from around 40% (sst1, sst2, sst4) to 67% (sst3, sst5) reflecting to some extent differences in receptor density (Bmax values published in accompanying paper, this journal). SRIF28 was slightly more potent than SRIF14 to inhibit FSAC at all five receptors, although the potency of the natural peptides SRIF14, SRIF28 and CST17 was generally similar with pEC50-values ranging from 7.5 to 8.7 depending on receptor and peptide. At SRIF1 receptors (sst2, sst3, sst5) most of the peptide analogues displayed full agonism (with some exceptions e.g. BIM 23056 at sst1-3 and sst5 receptors, and L362,855 and cycloantagonist SA at sst3 receptors), whereas at SRIF2 receptors these analogues tended to behave as partial agonists. BIM 23056 was an antagonist at sst3 receptors (antagonist binding constant pKB = 6.33), but not at other receptors. The AC inhibition profiles of sst1-5 receptors were compared with the different radioligand binding profiles as well as with [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding profile for sst2-5 receptors. High correlations were observed between FSAC inhibition, radioligand binding and [35S]GTPgammaS binding profiles at sst3, sst4 and sst5 receptors; by contrast, correlation coefficients at sst1 and sst2 receptors were low, and the binding profiles of [125I][Tyr10]CST14 correlated poorly. In line with these findings, the FSAC inhibition and [35S]GTPgammaS binding correlated poorly at sst2 receptors (sst1 receptors show no significant induction of [35S]GTPgammaS binding). The apparent lack of, or only weak, relationship between FSAC, radioligand or [35S]GTPgammaS binding observed for some SRIF receptors suggests that different active states may exist for these receptors, which may favour one transduction cascade over others.     

Characterisation of human recombinant somatostatin receptors. 2. Modulation of GTPγS binding

G protein activation by somatostatin (somatotropin release inhibiting factor, SRIF), cortistatin (CST) and analogues of these neuropeptides was investigated at human somatostatin receptor subtypes 1-5 (sst1-5) stably expressed in CCL39 Chinese hamster lung fibroblast cells by measuring agonist-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. [35S]GTPgammaS binding was assessed in the presence of 100 mM NaCl and 1 microM GDP, although higher Emax and/or pEC50 values may have been obtained under other conditions, but at the expense of lower absolute stimulation or signal/noise ratio. SRIF14 stimulated [35S]GTPgammaS binding to 162, 220, 148 and 266% of control levels via sst2, sst3, sst4 and sst5 receptors, respectively. At sst1 receptors, SRIF14 produced only a limited stimulation (Emax 115%). Hence sst1 receptors were not subjected to further [35S]GTPgammaS binding experiments. [35S]GTPgammaS binding assays were then performed with sst2-5 receptors. Most of the peptide analogues stimulated [35S]GTPgammaS binding in sst2-5 receptor-expressing cells. BIM 23056 behaved as an antagonist on SRIF14-induced [35S]GTPgammaS binding with an apparent pKBs of 6.33 and 5.84 at hsst3 and hsst5 receptors respectively, whereas neither agonism nor antagonism could be shown (at 1 microM) at sst2 or sst4 receptors. The effect at sst5 receptors was not surmountable and needs further investigations. The so-called "antagonist" SA, was devoid of antagonist activity at sst2 or sst3 receptors, whereas it was almost a full agonist at sst4 and sst5 receptor-mediated [35S]GTPgammaS binding. The [35S]GTPgammaS-binding profiles of hsst2-5 receptors were compared with their respective radioligand binding profiles. For sst4 and sst5 receptors, the rank order of affinity of all tested radioligands correlated highly significantly with [35S]GTPgammaS binding (r = 0.814-0.897). At sst3 receptors, [35S]GTPgammaS binding correlated somewhat less with binding profiles obtained with [125I][Tyr10]CST14 and [125I]CGP 23996 than with [125I]LTT-SRIF28 (r = 0.743, 0.757 and 0.882, respectively). At sst2 receptors, [35S]GTPgammaS binding correlated with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I][Tyr3]octreotide binding profiles (r = 0.596-0.699), but not with [125I][Tyr10]CST14 binding. The present [35S]GTPgammaS binding data combined to previous radioligand binding results obtained in cells expressing human SRIF receptors, suggest that at any given receptor, agonists' rank orders of potency (not to mention absolute affinity values which vary profoundly) are not as strictly ordered as may be anticipated. We are investigating these aspects further by analysing additional signalling pathways.     

Distribution of genotypes and response to alpha-interferon in patients with hepatitis C virus infection in Germany

To determine the distribution of hepatitis C virus (HCV) genotypes in German isolates, nucleotide sequences of the viral nonstructural 5 (NS5) genome domains were analyzed in isolates from 107 chronically HCV-infected patients. Of these 107 patients, 46 (43.0% were infected with subtype 1a and 47 (43.9%) with subtype 1 b. Six patients (5.6%) with a history of intravenous drug abuse were infected with subtype 3a. Eight patients (7.5%) who had acquired their HCV infection in Egypt carried subtype 4a. Forty-three of the 107 patients were treated with -interferon. Of these 43 patients, 16 (37.2%) were infected with subtype 1a and 27 patients (62.8%) with subtype 1b. Three patients infected with HCV-subtype 1a (18.7%) and four patients infected with subtype 1b (14.8%) showed a sustained complete response after interferon therapy. The HCV genotype 1 with its subtypes 1a and 1 b was the most common source of HCV infection in this group of patients. There was no significant difference in response to -interferon treatment of HCV infection with the subtypes 1a or 1b.     

Somatostatin receptors in the Rhesus monkey brain: localization and pharmacological characterization

To characterize the nature and distribution of somatostatin (SRIF) receptors, radioligand binding studies and in vitro receptor autoradiography were performed in Rhesus monkey brain using either [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) alone or in the presence of 3 nM seglitide (to block sst₂ sites), [¹²⁵1]Tyr³-octreotide or [¹²⁵I] CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) in buffer containing either 120 mM Na⁺ or 5 mM Mg²⁺. [¹²⁵I]Tyr³-octreotide labelled an apparently homogeneous population of sites in cerebral and cerebellar cortex (B _max = 27.3±2.8 fmol/mg protein and 52.6±8.6 fmol/mg protein, pK_d = 9.46±0.03 and 9.93±0.03, respectively). The pharmacological profile of these sites correlated highly significantly with that of human recombinant sst₂ receptors (r = 0.996), but not or much less with that of human recombinant sst₃ and sst₅ receptors (r = 0.12 and 0.45, respectively). [¹²⁵I]CGP 23996 (in Na⁺-buffer) also labelled an apparently homogeneous population of sites in Rhesus monkey cerebral cortex membranes (B _max = 3.1±0.3 fmol/mg protein, pKd = 10.57±0.08), the pharmacological profile of which was highly significantly correlated with the profiles of human recombinant sst₁ and sst₄ receptors (r = 0.98 and 0.96, respectively).Using receptor autoradiography, high levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide recognition sites were found in basal ganglia, molecular and granular layers of the cerebellum and layers III, V and VI of entorhinal cortex. In these regions, the addition of 3 nM seglitide produced a marked decrease of [¹²⁵I]LTT-SRIF-28 binding. Low levels of [¹²⁵I]LTT-SRIF-28 binding were observed in subiculum, pituitary and choroid plexus. By contrast, [¹²⁵I]CGP 23996 labelling in the presence of Mg²⁺ as well as Na⁺ ions was highest in pituitary and choroid plexus. However, [¹²⁵I]CGP 23996 binding was diversely affected by these ionic conditions in several regions of hippocampus and cerebral cortex. Displacement of [¹²⁵I]CGP 23996 (in Mg²⁺-buffer) with seglitide in the molecular layer of the cerebellum, deep layers of the entorhinal cortex, layers I, II and V of the insular cortex and frontal pole yielded complex competition curves suggesting the presence of two populations of SRIF receptors. By contrast, [¹²⁵I]CGP 23996 binding (in Mg²⁺-buffer) in the choroid plexus, hilus of the dentate gyrus and stratum oriens and radiatum of the CA3 field of hippocampus was not affected by seglitide up to 10 M, suggesting only sst₁ and/or sst₄ sites which have a negligible affinity for seglitide to be present in these structures.Taken together, these results suggest that [¹²⁵I]CGP 23996 (in the presence of Na⁺) labels exclusively SRIF-2 receptors (sst₁ and/or sst₄), whereas in the presence of Mg²⁺ ions, [¹²⁵I]CGP 23996 labels both SRIF-2 and SRIF-1 receptors (sst₂, sst₃ and sst₅). The present study also demonstrates the presence and differential distribution of sst₂ and sst₁/sst₄ receptors in the Rhesus monkey brain.     

Editor's note for debate Sporadic dementia of Alzheimer type: Role of amyloid in etiology is challenged

Summary Alzheimer's disease is a heterogenous neurodegenerative disorder. Whereas only a minority is due to genetic abnormalities and mostly with early onset, the majority of all Alzheimer cases is sporadic and with late onset. Therefore, in the latter, age-related disturbances in cellular metabolism may come into focus with respect to the etiopathogenesis rather than the primary formation of amyloid. In this Editor's note for debate, the role of amyloid as a causative factor of sporadic Alzheimer's disease is challenged. Instead, as a possible primary abnormal event in sporadic Alzheimer's disease, the perturbations in neuronal glucose metabolism and the subsequent ATP deficit with its impacts on the secondary amyloid formation are discussed to open a new field of research and another aspect for debate.     

Konstitutionsaufklärung der Reaktionsprodukte aus Benzonitriloxid und 1,4-disubstituierten Urazolen

Structural Elucidation of the Reaction Products from Benzonitrile Oxide and 1,4-Disubstituted Urazoles The reaction of benzonitrile oxide with 1,4-disubstituted urazoles 1 does not yield 1,4-disubstituted 3-(phenylcarbamyoloxy)--1,2,4-triazolin-5-ones 2 , as reported in the literature, but leads to N¹′,N³′-disubstituted 3-phenyl-4-ureido--1,2,4-oxadiazolin-5-ones 4 .     

Antipsychotic drugs and dopamine-mediated responses in Aplysia neurons

Summary The effect of antipsychotic drugs was tested on responses to micro-electrophoretically applied dopamine, acetylcholine and 5-hydroxy-tryptamine in identified neurons of the marine gastropod Aplysia californica. Fluphenazine was able to depress the response to DA in concentration of 10M, with 100M DA-responses of many neurons were blocked completely. Thioridazine (10 and 100M) and haloperidol (50M) were also effective in depressing DA-responses, while the non-antipsychotic phenothiazines mepazine (10 and 100M) and promethazine (100M) had only a slight action on DA-receptors. ACh-and 5-HT-responses were slightly affected only by high concentrations after long lasting perfusion. The investigated drugs had no persistent or only an insignificant effect on resting membrane potential and amplitude of action potentials of the neurons. With haloperidol depolarizing afterpotentials leading to double discharges were observed in some neurons. In a few instances spontaneous EPSPs disappeared with the DA-response under the influence of anti-psychotic drugs.The results render a direct neurophysiological evidence for the blockade of DA-receptors by antipsychotic drugs in correspondence to their clinical efficacy and agree with data from clinical observations and obtained in neurochemical, behavioral and indirect neurophysiological experiments.Supported by the österreichischen Fonds zur Förderung der wissenschaftlichen Forschung. —A preliminary report of a part of the results was published in Experientia30, 1318–1320 (1974).     

Letal verlaufende Varizellen nach Nierentransplantation

Zusammenfassung Wir berichten über ein 7 Jahre altes M?dchen, das 14 Tage nach einer Nierentransplantation an Varizellen erkrankte. Erste Symptome waren krampfartige Bauchschmerzen mit radiologischen Hinweisen auf einen Obturationssubileus bei Obstipation. Als nach abführenden Ma?nahmen keine Besserung auftrat, wurde eine Probelaparotomie mit Entfernung des Tenckhoff-Dialysekatheters durchgeführt. Dabei zeigte sich kein pathologischer Befund, insbesondere fanden sich keine Briden. Zwei Tage nach Beginn der abdominalen Symptomatik trat ein zun?chst uncharakteristisches Exanthem auf. Aus den sich in den n?chsten 12 h entwickelnden Bl?schen konnte Varicella-Zoster-Virus isoliert werden. Trotz hochdosierter Gabe von Aciclovir und Varicella-Zoster-Immunglobulin entwickelte sich ein fulminantes Leberversagen mit Verbrauchskoagulopathie. Die Patientin verstarb am 18. postoperativen Tag trotz intensiver supportiver Therapie im Multiorganversagen. Diskussion: Vor dem Hintergrund dieser letalen Varizelleninfektion wird auf die Komplikationen und die teilweise untypischen Verl?ufe von Windpocken bei immunsupprimierten Kindern hingewiesen. Eine aktive Impfung ist daher bei seronegativen Patienten vor einer geplanten Organtransplantation zu fordern.      

Hb Manukau [β67(E11)Val → Gly; HBB: c.203T>G]: The Role of Genetic Testing in the Diagnosis of Idiopathic Hemolytic Anemia

The increasing availability of DNA sequencing of globin genes has improved our ability to detect conditions that were presumed to be extremely rare. These conditions may remain undiagnosed due to unfamiliarity with clinical presentation, relative unavailability of advanced diagnostic alternatives, or may defy detection by being electrophoretically silent or extreme instability rendering their presence to be below detection level. Genetic studies were pursued in a mother and daughter with severe hemolytic anemia as initial testing failed to be diagnostic. DNA sequence analysis of the β-globin gene identified Hb Manukau [β67(E11)Val?→?Gly; HBB: c.203T?>?G], an extremely unstable hemoglobin (Hb) variant. This is the second family described with this condition (first in the western hemisphere). An astute clinician may benefit from being persistent and pursuing additional testing including molecular genetic characterization where clinical suspicion remains high.     

Screening for Depression in Emergency Department Patients

Background: Depression is a common disease, yet it is not commonly studied in the Emergency Medicine literature. Study Objectives: To evaluate the prevalence of emergency department (ED) patients who have the symptoms of depression. Design: This was a prospective observational study performed at two EDs over a 9-month period. Adult patients were screened for depression symptoms by Diagnostic and Statistical Manual of Mental Disorders, 4^th edition criteria. Results: There were 505 patients screened from April through December, 2004. Of the 505 patients, 109 (21.6%) screened positive for the symptoms of depression. The prevalence of positive screens was similar at each ED. Conclusion: About 1 in 5 ED patients may be suffering with depression.