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BackgroundDiagnosis of TB in pediatric population poses several challenges. A novel initiative was implemented in several major cities of India aimed at providing upfront access to free-of-cost Xpert MTB/RIF to presumptive pediatric TB cases. This paper aims to describe the experience of implementing this large initiative and assess feasibility of the intervention in high TB burden settings.MethodsData were drawn from the pediatric TB project implemented in 10 major cities of India between April 2014 and March 2018. In each city, providers, both public and private, were engaged and linked with a high throughput Xpert MTB/RIF lab (established in that city) through rapid specimen transportation and electronic reporting system. Rates and proportions were estimated to describe the characteristics of this cohort.ResultsOf the total 94,415 presumptive pediatric TB cases tested in the project, 6,270 were diagnosed positive for MTB (6.6%) on Xpert MTB/RIF (vs 2% on smear microscopy). Among MTB positives, 545 cases were rifampicin resistant (8.7%). The median duration between collection of specimens and reporting of results was 0 days (same day) and >89% cases were initiated on treatment. Approximately 50% of the specimens tested were non-sputum. The number of providers/facilities engaged under the project increased >10-fold (from 124 in Q2'14 to 1416 in Q1'18).ConclusionThis project, which was one of the largest initiatives globally among pediatric population, demonstrated the feasibility of sustaining rapid and upfront access to free-of-cost Xpert MTB/RIF testing. The project underscores the efficiency of this rapid diagnostic assay in tackling several challenges in pediatric TB diagnosis, identifies opportunities for further interventions as well as brings to light scope for effective engagement with healthcare providers. The findings have facilitated a policy decision by National TB Programme mandating the use of Xpert MTB/RIF as a primary diagnostic tool for TB diagnosis in children, which is being scaled-up.

Neuronal nicotinic acetylcholine receptor (nAChR) agonists active at the alpha-7 (α-7) receptor subtype are potential therapeutics for cognitive deficits in schizophrenia, Alzheimer's disease, and other mental disorders. SSR180711, an α-7 selective partial agonist, has been shown to improve preclinical cognition. A novel positron emission tomography (PET) radioligand, ¹¹C-Chiba1001, is a close analog of SSR180711. We labeled Chiba-1001 with tritium in order to evaluate its utility as a preclinical radioligand tool. In vitro, the binding affinity of [³H]Chiba-1001 at the α-7 receptor was low (K(d) = 120-180 nM) in both HEK239 cell membranes expressing human α-7 receptor and in native rat hippocampus membranes. The α-7 selective ligands AZD0328, ARR17779, and MLA did not inhibit [³H]Chiba-1001 binding (K(i) > 10,000 nM). In rat hippocampal membranes, Chiba-1001 and SSR180711 inhibited [³H]Chiba-1001 binding (K(i) = 220 and 230 nM, respectively), consistent with the literature reports. The in vivo binding profile of the radioligand was examined in normal rat, wild type mouse, and α-7 knockout mouse brain. We found that [³H]Chiba-1001 lacks adequate and specific brain regional uptake in rat and mouse brain. No significant inhibition of the radioligand binding was obtained following pretreatment of the animal with AZ11637326, AZD0328, or MLA. Our results indicate that [³H]Chiba-1001 has low affinity for α-7 nAChRs in vitro and poor α-7 regional and pharmacological selectivity in the rodent brain.

Recent reports have indicated that patients with schizophrenia have a profound hypo-functionality of glutamatergic signaling pathways. Positive allosteric modulation of mGlu(5) receptor has been postulated to augment NMDA function and thereby alleviate the glutamatergic hypo-function observed in schizophrenic patients. Here we report the in vitro and in vivo characterization of CPPZ (1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone), a structurally novel positive allosteric modulator selective for mGlu(5) receptor. In HEK293 cells stably over-expressing human mGlu(5) receptor, CPPZ potentiates the intracellular calcium response elicited by a suboptimal concentration of the endogenous agonist glutamate. CPPZ does not have any intrinsic agonist activity and behaves functionally as a positive allosteric modulator. This is further supported by binding data, which demonstrate that CPPZ is able to displace the negative allosteric modulator MPEP but does not compete with the orthosteric ligand quisqualic acid. Instead, CPPZ enhances the binding of the orthosteric ligand. In native preparations, CPPZ potentiates calcium flux in rat cortical neurons stimulated with the group I agonist dihydroxyphenylglycine (DHPG). In addition, CPPZ modulates long-term potentiation in rat hippocampal slices, a process known to be NMDA dependent. In vivo, CPPZ reverses hyper locomotion triggered by the NMDA open channel blocker MK801 in CD1 mice. CPPZ was also able to reduce rat conditioned avoidance responding to electric shock. Both in vitro and in vivo data demonstrate that this novel compound acts as an mGlu(5) receptor positive allosteric modulator, which modulates NMDA dependent responses and suggests that the enhancement of mGlu(5) receptor activity may prove useful in the treatment of schizophrenia.

Positive allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) is regarded as a potential novel treatment for schizophrenic patients. Herein we report the synthesis and SAR of 4-aryl piperazine and piperidine amides as potent mGluR5 positive allosteric modulators (PAMs). Several analogs have excellent activity and desired drug-like properties. Compound 2b was further characterized as a PAM using several in vitro experiments, and produced robust activity in several preclinical animal models.

New analogues of the previously described 3-aryl pyridone KOR agonists have been synthesised by parallel synthetic methods, both in solution- and with solid-phase chemistry, making use of the well known and versatile Mitsunobu, Suzuki and Buchwald reactions. Opioid receptor binding data for the compounds produced is reported.

A new series of 3-aryl pyridone based kappa opioid receptor agonists was designed and synthesised, based on an understanding of the classical kappa opioid receptor pharmacophore. The most potent of the new compounds were comparable to U-69,593 in receptor affinity, selectivity and functional agonist effect at the cloned human kappa opioid receptor.

To delineate domains essential for G-protein coupling in melanocortin 1 receptor (MC1R), we mutated polar and basic residues to alanine at eleven positions in the putative third intracellular loop and determined consequent changes in the ligand binding and generation of second messenger cAMP. Results demonstrate that ligand binding affinity was not affected by any of the mutations. However, every mutant displayed reduced functional response as compared to the wild type receptor. Replacement of residues (K226, R227, Q228, R229, H232, Q233 and K238) present in second half of third intracellular loop resulted in an almost complete loss of functional response. The results have demonstrated that the amino acid residues present in C-terminal portion of third intracellular loop of MC1R are involved in coupling to G-protein and that a region of four amino acids, K226-R227-Q228-R229 is essential for coupling of MC1R to G-protein.

The human melanocortin 5 receptor (hMC5R) in the melanocortin receptor family has been identified as the receptor with low affinity towards alpha-MSH. Here we show that the glutamine at position 235 and arginine at the position 272 in the hMC5R are contributing to the low affinity of this receptor. Glutamine235 and arginine272 in hMC5R were mutated to lysine (Q235K) and cysteine (R272C), respectively, residues which are conserved at these positions in other melanocortin receptor subtypes. Upon these mutations affinity of alpha-MSH for hMC5R was increased 10-fold for Q235K and 690-fold for R272C mutants, respectively. The results explain the unusually low affinity of the hMC5R to the melanocortic ligands and suggest the importance of these conserved residues in maintaining the high affinity form of melanocortin receptors.

To investigate whether residues in the extracellular domains of melanocortin 1 receptor (MC1R) are required for ligand binding, a number of mutants were constructed where charged residues were converted to alanine. The residues targeted for mutagenesis were Ser6, Glu102, Arg109, Asp184, Glu269, and Thr272. The mutant receptor DNAs were transiently expressed in COS-1 cells and their ability to bind [N1e4,D-Phe7]-alpha-MSH (NDP-MSH) was evaluated. Substitution of Asp184 by alanine completely abolished the binding of radiolabelled NDP-MSH as well as ACTH, even though the mutated receptor could be detected on cell surface using anti MC1R specific polyclonal antiserum. Mutations of Ser6, Glu269 and Thr272 resulted in a considerable loss of affinity for radiolabelled NDP-MSH as well as the ability of alpha-MSH to displace the bound radiolabelled NDP-MSH. The results demonstrate that the extracellular loops of human MC1R contain important ligand binding epitopes.

The localization of the melanocortin 1 (MC1) receptor in brain was investigated by in situ hybridization and immunohistochemistry. In rat periaqueductal gray matter (PAG), a small number of cells which displayed a punctate distribution showed a specific hybridization signal for MC1 receptor mRNA, whereas other regions studied were negative. In human PAG, the immunoreactivity for MC1 receptor was detected in scattered cells. These results indicate that a restricted distribution of MC1 receptor is present in the central nervous system.

Peptides of 11 and 15 residue lengths were synthesised according to the sequence of the N-terminal region of the human MC1 melanocyte stimulating hormone receptor. The peptides were conjugated to thyroglobulin and used for preparation of antisera in the rabbit. Each of the conjugates raised antisera which showed high titre and specificity for its respective peptide antigen when evaluated in an ELISA test. Both types of antisera immunostained MC1 receptor expressing COS-7 cells. By contrast, the sera did not stain control COS-7 cells not expressing the MC1 receptor. Moreover, preimmune sera or antiserum preadsorbed with its respective peptide did not stain the MC1 receptor expressing cells. The antisera were used to immunostain sections of normal human skin, as well as samples of cutaneous malignant melanoma tumours obtained from a patient. The cells of the melanoma tumours were very strongly immunostained with the MC1 receptor antisera. By contrast, melanocytes which were present in the normal skin could not be visualised with our antisera.

The DNAs encoding three melanocortin receptor subtypes (melanocortin MC1 receptor, melanocortin MC3 receptor and melanocortin MC5 receptor) were expressed individually in COS (CV-1 Origin, SV40) cells to characterise their ligand binding properties. The results indicated that [125I][Nle4, D-Phe7]alpha-MSH (melanocyte stimulating hormone) bound to a single saturable site with Kd values of 85.1 +/- 8.0 pmol/l (mean +/- S.E.M), 396 +/- 65 pmol/l and 5.05 +/- 1.00 nmol/l for melanocortin MC1 receptor, melanocortin MC3 receptor and melanocortin MC3 receptor, respectively. The melanocortin MC1 receptor and the melanocortin MC5 receptor showed a similar potency order to the melanocortic peptides examined which was markedly different from the potency order of the melanocortin MC3 receptor. The melanocortin MC1 receptor and melanocortin MC5 receptor had a relatively higher affinity for alpha-MSH than gamma-MSH and beta-MSH, whereas the melanocortin MC3 receptor had higher affinity for desacetyl-alpha-MSH, gamma-MSH and beta-MSH compared to alpha-MSH. The inclusion of the endopeptidase inhibitor phosphoramidon to prevent the breakdown of ACTH-(1-39) (adrenocorticotrophic hormone) to alpha-MSH, decreased ACTH-(1-39) binding affinity showing that ACTH-(1-39) had a much lower affinity for melanocortin MC1 receptor than reported earlier.

The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu5) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu5 compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu5 and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC50 of 87 nM with an 89% effect in transfected HEK293 cells and an EC50 of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu5 in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [3H]MPEP, the mGlu5 receptor negative allosteric modulator (NAM), PBPyl exhibited a k i value of 34 nM. PBPyl also displaced [3H]ABP688, a mGluR5 receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu5b membranes, [3H] PBPyl exhibited saturable binding with a Kd value of 18.6 nM. In competition binding experiments, [3H] PBPyl was displaced by high affinity mGlu5 positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu5 receptor PAM, this study offers value toward the development of novel PET imaging agents for this important therapeutic target. Synapse, 2013. © 2012 Wiley Periodicals, Inc.

The molecular basis for the alpha-melanocyte stimulating hormone (alpha-MSH) stereoselectivity was studied by examining ligand binding to site specific mutants of the melanocortin 1 receptor (MC1R). The amino acids Asp117, Phe179, His209 and His260 were targeted for mutation to alanine as they are conserved in the melanocortin receptor family. Expression of the wild type and the MC1R mutants in COS-7 cells revealed that the binding affinities for the alpha-MSH (L-isomer) was reduced by 267 fold for the D117-->A mutant and 132 fold for the H260-->A mutant. In contrast, the binding affinity for the [Nle4, D-Phe7]-alpha-MSH (NDP-MSH; D-isomer) remain unchanged between the wild type and the mutants. Moreover, the mutants also displayed reduction in affinity to L-isomers of all the other melanocortic peptides examined. Thus, the mutation of single amino acids in the third and sixth transmembrane segments results in the display of ligand stereoselectivity of the MC1R. In addition, these data represent the first evidence of the different binding epitopes on a G-protein coupled receptor for a peptide ligand stereoisomers, both of which are shown to be potent agonists.

Analysis of saturation isotherms of the novel α2-adrenoceptor antagonist radioligand [3H]-MK 912 revealed that the ligand labelled a homogenous population of α2B-adrenoceptors in the neonatal rat lung with a Kd of 0.77 nM and a Bmax of 231 fmol/mg protein. In rat kidney, combined saturation and competition experiments, using [3H]-MK 912 and the α2A-adrenoceptor selective drug guanfacine, revealed that ~ 81% of the sites labelled by [3H]-MK 912 were α2B-adrenoceptors and ~ 19% α2A-adrenoceptors; the Kds of [3H]-MK 912 being. 1.1 and 2.0 nM and the Bmax 134 and 33 fmol/mg protein, respectively. The kidney α2B-adrenoceptors were studied separately by using ~ 1.5 nM [3H]-MK 912 in the presence of 0.32 μM guanfacine, the latter which blocked ligand binding to α2A-adrenoceptors completely. Analysis of drug competition curves obtained during these conditions revealed that 18 out of 20 different agonists and antagonists yielded steep and uniphasic competion curves which modelled best into one site fits. However, both guanoxabenz and LT 11 appeared to inhibit [3H]-MK 912 binding at two sites; the Kds of guanoxabenz differing ~ 120-fold and that of LT 11 differing ~ 35-fold for the two sites. Moreover, the addition of mutual fixed concentrations of either 20 μM guanoxabenz or 20 μM LT 11 completely prevented the binding of the other compound to its high affinity site, indicating that both compounds labelled the same site with the high affinity. The analysis indicated that 29% of the sites were of high and 71% of low affinity. However, in the neonatal rat lung guanoxabenz and LT 11 (as well as 15 other compounds) yielded competition curves which modelled only into one site fits. The Kds obtained in the lung correlated well with the Kds obtained in the kidney for α2B-adrenoceptors; for guanoxabenz and LT 11 the values from the lung were close to those determined in the kidney for the low affinity site for guanoxabenz and LT 11. Moreover, when the rat RNG α2B-adrenoceptor was expressed in COS-7 cells and its binding properties tested using [3H]-MK 912 binding, guanoxabenz, LT 11 as well as a number of other drugs inhibited the ligand binding at a single α2-adrenoceptor site; the drug Kds being practically the same as those found for the neonatal rat lung. It is suggested that rat α2B-adrenoceptors may exist in two forms: α2B1 and α2B2. The α2B1-form, which shows high affinity for guanoxabenz and LT 11, is present in the adult rat kidney. The α2B2-form, which shows low affinity for guanoxabenz and LT 11, is present in the neonatal rat lung as well as in the adult rat kidney and it is encoded by the RNG gene.

Neuronal nicotinic acetylcholine receptor (nAChR) agonists active at the alpha-7 (α-7) receptor subtype are potential therapeutics for cognitive deficits in schizophrenia, Alzheimer's disease, and other mental disorders. SSR180711, an α-7 selective partial agonist, has been shown to improve preclinical cognition. A novel positron emission tomography (PET) radioligand, ¹¹C-Chiba1001, is a close analog of SSR180711. We labeled Chiba-1001 with tritium in order to evaluate its utility as a preclinical radioligand tool. In vitro, the binding affinity of [³H]Chiba-1001 at the α-7 receptor was low (K(d) = 120-180 nM) in both HEK239 cell membranes expressing human α-7 receptor and in native rat hippocampus membranes. The α-7 selective ligands AZD0328, ARR17779, and MLA did not inhibit [³H]Chiba-1001 binding (K(i)10,000 nM). In rat hippocampal membranes, Chiba-1001 and SSR180711 inhibited [³H]Chiba-1001 binding (K(i) = 220 and 230 nM, respectively), consistent with the literature reports. The in vivo binding profile of the radioligand was examined in normal rat, wild type mouse, and α-7 knockout mouse brain. We found that [³H]Chiba-1001 lacks adequate and specific brain regional uptake in rat and mouse brain. No significant inhibition of the radioligand binding was obtained following pretreatment of the animal with AZ11637326, AZD0328, or MLA. Our results indicate that [³H]Chiba-1001 has low affinity for α-7 nAChRs in vitro and poor α-7 regional and pharmacological selectivity in the rodent brain.

We previously reported the absence of high-affinity binding of the group II metabotropic glutamate receptor agonists LY 354,740 and LY 379,268 to the D2L dopamine receptor. A rebuttal to our findings has since been reported (see Introduction section); this study represents our response. Analysis by LCMS of LY 354,740 and LY 379,268 used in this study revealed the correct molecular mass for these compounds. Both LY 354,740 and LY 379,268 exhibited potent agonist activity for mGluR2 in the 35S-GTPγS assay. Functionally, neither compound displayed antagonist activity in the GTPγS assay with recombinant D2. At concentrations up to 10 μM, both compounds failed to displace [3H]-raclopride, [3H]-PHNO, or [3H]-domperidone in filter-binding assays under isotonic (120 mM NaCl or N-methyl glucamine) or low-ionic strength (no NaCl or N-methyl glucamine) conditions. Some displacement of [3H]-domperidone (20–40%) was observed at 30 μM of LY 354,740 under low-ionic strength and under isotonic conditions in the absence of NaCl. No displacement of [3H]-domperidone was detected in a two site model at lower (

Metabotropic glutamate receptors (mGluRs), which are coupled to second messenger pathways via G proteins, modulate glutamatergic and GABAergic neurotransmission. Because of their role in modulating neurotransmission, mGluRs are attractive therapeutic targets for anxiety disorders. Previously we showed that mGluR8(-/-) male mice showed higher measures of anxiety in the open field and elevated plus maze than age-matched wild-type mice. In this study, we assessed the potential effects of acute pharmacological modulation of mGluR8 on measures of avoidable and unavoidable anxiety. In addition to wild-type mice, we also tested apolipoprotein E-deficient (Apoe(-/-)) mice, as these mice show increased levels of anxiety-like behaviors and therefore might show an altered sensitivity to mGluR8 stimulation. mGluR8 stimulation with the specific agonist DCPG, or modulation with AZ12216052, a new, positive allosteric modulator of mGluR8 reduced measures of anxiety in both wild-type mice. The effects of mGluR8 positive allosteric modulators, which only affect neurotransmission in the presence of extracellular glutamate, seem particularly promising for patients with anxiety disorders showing benzodiazepine insensitivity.

Melanocytes and melanoma cells are known to possess receptors for melanocyte stimulating hormone (MSH). A cDNA clone, designated 11D, has been isolated from human melanoma cells and encodes a MSH receptor. The cloned cDNA encodes a 317 amino acid protein with transmembrane topography characteristics of a G-protein-coupled receptor, but it does not show striking similarity to already published sequences of other G-protein-coupled receptors. When 11D cDNA is expressed in COS-7 cells, it binds an 125I-labelled MSH analogue (NDP-MSH) in a specific manner. The bound ligand could be displaced by melanotropic peptides, alpha-MSH, beta-MSH, gamma-MSH and ACTH (adrenocorticotropic hormone), but not by the non-melanotropic peptide, beta-endorphin. This is the first report of the cloning of the receptor gene of the melanotropin receptor family.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. ( Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The K i (stoichiometry of the enzyme—antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.

alpha-Melanocyte stimulating hormone (alpha-MSH) regulates skin and hair pigmentation by modulating the activity of MSH receptor (MC1R). We have identified Arg151Cys variant of human MC1R in genomic DNA isolated from a person with red hair and light skin of type I. The Arg151Cys variant of MC1R binds to radio-labelled analogue of alpha-MSH with identical affinity as wild type MC1R but can not be stimulated to produce cyclic AMP (cAMP). The mutation Arg151Cys renders human MC1R completely nonfunctional, which explains the red hair, light skin and poor tanning ability (skin type I). This is the first report ever describing a nonfunctional MC1R isolated from a human subject.

This study was conducted to determine the binding properties of recently discovered, putative alpha-MSH antagonist 153N-6 peptide at melanocortin receptor subtypes. The results indicated that 153N-6 peptide can competitively inhibit [125I]NDP-MSH binding from all the receptor subtypes investigated. The relative potency order of 153N-6 for inhibiting [125I]NDP-MSH binding was MC1R (Ki 955 +/- 35.7 nM) = MC4R (Ki 1151 +/- 106 nM) > MC3R (Ki 3229 +/- 637 nM) > MC5R (Ki 6286 +/- 462 nM), which is different than the potency order of either NDP-MSH or alpha-MSH. Substitution of aspartic acid117 and histidine260 by alanine in melanocortin 1 receptor resulted in a 4.75-fold decrease (Ki 4541 +/- 644 nM) and an 11-fold increase (Ki 84.29 +/- 4.53 nM), respectively, in the relative potency of 153N-6 for competitively inhibiting [125I]NDP-MSH binding.

Neuropeptide Y (NPY), a 36-amino-acid peptide widely expressed in the brain is involved in many physiological responses, including hypothalamic control of food intake and cardiovascular homeostasis. NPY mediates its effects through binding to the Y1, Y2 and Y5 G-protein-coupled receptors. Little is known of the role of the Y2 receptor in mediating the different NPY effects. We inactivated the Y2 receptor subtype in mice and found that these mice developed increased body weight, food intake and fat deposition. The null mutant mice showed an attenuated response to leptin administration but a normal response to NPY-induced food intake and intact regulation of re-feeding and body weight after starvation. An absence of the Y2 receptor subtype also affected the basal control of heart rate, but did not influence blood pressure. These findings indicate an inhibitory role for the Y2 receptor subtype in the central regulation of body weight and control of food intake.

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.

Corticotropin is produced by keratinocytes and may have an immunoregulatory role in oral mucosa and skin. We have investigated its effects on a human oral keratinocyte cell line and shown that corticotropin, acting via its specific receptor, stimulates a dose-dependent increase in DNA synthesis and induces cell proliferation. When cells were incubated in the presence of increasing concentrations of corticotropin, there were significant increases in intracellular cAMP levels. Corticotropin-stimulated mitogenesis and cell proliferation were attenuated by the adenylyl cyclase inhibitor SQ22,536, but were unaffected by inhibitors of protein kinase C or tyrosine kinase. These data identify corticotropin as a mitogenic regulatory peptide of keratinocytes acting via cAMP.

We describe the immunohistochemical detection of the melanocortin 1 receptor (MC1R) protein in human gonadal tissues using a specific monoclonal antibody. The MC1R was found to be present in Leydig's cells in testis, in lutein cells in the corpus luteum and in the nucleus of the trophoblastic cells of the placenta. Though it has been speculated earlier that MC1R is present in gonadal tissues, this is the first report demonstrating the presence of MC1R protein in these cells.

The mouse adrenocortical cell line Y1, that expresses ACTH receptors (MC2R), was used to probe the binding of ACTH and MSH peptides by using radio-labelled ACTH (1-39). The Y1 cells were found to bind [125I]-labelled ACTH (1-39) with high affinity (Kd approximately 130 pM). However, none of the melanocortin peptides NDP-MSH, alpha-MSH, beta-MSH or gamma 1-MSH could compete with the binding of the labelled ACTH(1-39). When other MC receptor subtype DNAs (MC1, MC3 and MC4) were transfected into the Y1 cells, characteristic binding of the [125I]NDP-MSH appeared for each of the receptor subtype, but no specific binding was present in non-transfected cells. This is the first report clearly demonstrating that the ACTH receptor binds only ACTH, but not other melanocortin peptides.

Stable expression of the MSH receptor in a homologous system is important for the study of the function and mechanism of signalling of this receptor. This is the first report on the stable expression of the human alpha-MSH receptor in the mouse melanoma G4F clone which lacks an endogenous MSH receptor. Several stable transfectant cell lines were obtained all of which express the human MSH receptor in high numbers. Human MSH receptor mRNA expression was detected by Northern blot analysis. Competition binding experiments showed that the MSH receptors expressed in these cells have the same affinity for [Nle4,D-Phe7]-alpha-MSH as the MSH receptors of the human HBL melanoma cell line. Several of the transfectant cell lines produced melanin constitutively, some of them secreting melanin into the medium whereas other clones did not secrete melanin. MSH and cholera toxin did not or only marginally increase melanogenesis in these clones, and forskolin had an opposite effect. These results suggest that the human MSH receptor may be constitutively active in these transfected mouse melanoma cells.

A human genomic clone designated MC-2 is isolated. The cloned DNA codes for a protein of 325 amino acids which possesses seven hydrophobic segments, a characteristic of G-protein coupled receptors. The MC-2 receptor is expressed in brain tissue but not in the melanoma cells. When the MC-2 DNA is expressed in COS-7 cells, it binds [125I]-labelled [Nle4, D-Phe7]- alpha melanocyte stimulating hormone (NDP-MSH) which then could be displaced by melanotropic peptides alpha-MSH, beta-MSH, gamma-MSH and adrenocorticotropic hormone, but not by non-melanotropic peptide beta-endorphin. The highest affinity of 5.18 nM was for the NDP-MSH peptide. The novel MC-2 receptor and the MC-1 receptor, described earlier by us (8) showed identical order of affinity for the melanocortin peptides, but the affinities and the fold differences in the affinities to the melanocortin peptides were different when compared to the earlier described MC-1 receptor. The results suggest that the MC-2 DNA codes for a novel melanocortin receptor.

Rat alpha 2B-adrenoceptor was expressed in Escherichia coli using 'ATG vector' containing cDNA encoding the 'non-glycosylated rat alpha 2-adrenoceptor' (RNG alpha 2). The highest receptor binding activity (using the alpha 2-adrenoceptor ligand [3H]MK 912) was found when transfected bacteria cultures were grown at 30 degrees C for about 4 h after induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Saturation experiments showed that the radioligand bound to a single saturable site with a Kd of 1.42 +/- 0.09 nM and capacity of 281 +/- 6 fmol/mg protein. Binding constants of 14 compounds for the rat alpha 2B-adrenoceptor expressed in E. coli were determined and compared to the values previously obtained for the rat alpha 2B-adrenoceptor when expressed in COS cells as well as for native neonatal rat lung alpha 2B-adrenoceptors. The results indicate that when the rat alpha 2B-adrenoceptor is expressed in E. coli it retains identical ligand binding properties to those found when the receptor is present in the eukaryotic system. Expressing alpha 2B-adrenoceptors in E. coli would, therefore, seem to constitute a valid alternative in, e.g., drug screening and structure analysis of the alpha 2B-adrenoceptors.

1. Simultaneous computer modelling of control and guanfacine-masked [3H]-MK 912 saturation curves as well as guanfacine competition curves revealed that the drugs bound to two alpha 2-adrenoceptor subtypes in the rat cerebral cortex with very different selectivities. These alpha 2-adrenoceptor subtypes were designated alpha 2A and alpha 2C. The Kd value of [3H]-MK 912 for the alpha 2A-subtype was 1.77 nM and for the alpha 2C-subtype 0.075 nM; the receptor sites showing capacities 296 and 33 fmol mg-1 protein, respectively. The Kds of guanfacine were 19.9 and 344 nM, respectively. 2. Binding constants of 26 compounds for the two rat cerebral cortex alpha 2-adrenoceptor subtypes were determined by simultaneous computer modelling of control and guanfacine-masked drug competition curves as well as plain guanfacine competition curves using [3H]-MK912 as labelled ligand (i.e. a '3-curve assay'). Of the tested drugs WB4101, corynanthine, rauwolscine, yohimbine, ARC 239 and prazosin were found to be clearly alpha 2C-selective with selectivities ranging from 16 to 30 fold whereas guanfacine, oxymetazoline, BRL 44408 and BRL 41992 were found to be alpha 2A-selective with selectivities ranging from 9 to 22 fold. 3. The Kds of compounds obtained for the cerebral cortex alpha 2C-adrenoceptors showed an almost 1:1 correlation with the corresponding Kds for alpha 2-adrenoceptors expressed by the pA2d-gene (the rat 'alpha 2-C4' adrenoceptor) in CHO-cells. The cerebral cortex alpha 2A-adrenoceptors did not correlate well with the pA2d alpha 2-adrenoceptor Kds. 4. In the rat spinal cord [3H]-MK 912 bound to alpha 2A- and alpha 2C-adrenoceptor sites with similar affinities as in the cerebral cortex and with densities 172 and 7.4 fmol mg-1 protein, respectively. Drug affinities for some compounds showing major selectivity for alpha 2A- and alpha 2C-adrenoceptors were fully compatible with the notion that the spinal cord sites were alpha 2A- and alpha 2C-adrenoceptors.

Two forms of the smaller subunit of the human soluble guanylate cyclase enzyme have been cloned by using PCR. One of the clones (HSGC-1) is identical to bovine and rat lung smaller subunit cyclase. However, the other (HSGC-2) is lacking 33 amino acids. Comparison of its sequence with published partial genomic sequences of bovine guanylate cyclase indicates that HSGC-2 is formed due to alternative splicing.

A number of imidazoline, imidazole and guanidinium compounds and other drugs were compared for their ability to bind to non-adrenergic idazoxan (imidazoline) I-receptors in particulate guinea pig cerebral cortex and ileum smooth muscle fractions. Radioligand binding with [ 3 H]idazoxan was used for the experiments. Computer modelling of the binding data gave dissociation constants for drug binding to both I-receptors and α 2 -adrenoceptors. Most drugs showed similar affinities for I-receptors in cortex and ileum. However, medetomidine stereoisomers as well as a few other drugs clearly delineated the I-receptors in cortex and ileum as different.

A single step immunoaffinity purification procedure for human erythrocyte acetylcholinesterase is described which permitted the isolation of milligram quantities of enzyme from 10 U of erythrocytes, with 113 000-fold purification and a yield of about 22%. In SDS-PAGE analysis, the enzyme corresponds to a disulfide linked dimer of 140 kDa which is converted to a 70 kDa monomer upon disulfide reduction. The tryptic peptides generated from purified enzyme were separated by reverse-phase HPLC. Five of these peptides were analysed to determine the amino acid sequences. The obtained sequences showed no homology to the already known amino acid sequences for human serum and brain butyryl-cholinesterase and Torpedo californica acetylcholinesterase.

The 37 K protein, earlier found to be present in 3.75% PEG-precipitates from sera of untreated patients with CML, was further characterized. Gel filtration at neutral pH resolved the PEG-precipitate into a non-IgG containing protein peak-I and a IgG containing peak-II. Immunoprecipitation of peak-II with antihuman IgG antiglobulin and subsequent 2D-SDS-PAGE analysis of the immunoprecipitate revealed the presence of 37 K protein in peak-II confirming its association with IgG. 125 I-37 K protein was found to interact with antibodies isolated from autologous and allogenic CML-CIC samples but not with similarly isolated antibodies from normal subject, and patients with AML, ALL, MF, and HD. Peptide maps generated by tryptic digestion of 37 K protein (from five different CML patients) were found to be identical. Specific interaction of 37 K protein with the autologous and allogenic antibodies and identity of peptide maps lead to the conclusion that the 37 K protein is a CML-associated antigen appearing in CIC.

A 3.75% polyethylene glycol -6000 mediated turbidimetric method was used for the estimation of levels of circulating immune complexes (CIC) in sera of normal human subjects and patients with chronic myeloid leukemia (CML). In terms of equivalents of heat-aggregated gamma globulin (eq.HAGG mg/ml serum), the mean levels of CIC were: 5.69 mg/ml (SD 4.78) in 81 normal human subjects; 22.63 mg/ml (SD 9.86) in 52 untreated CML patients; 7.61 mg/ml (SD 5.74) in 54 CML patients in remission; and 21.70 mg/ml (SD 8.15) in 18 CML patients in relapse. High CIC levels, thus, showed a significant association with the pretreatment -- and relapse -- status of the disease (p less than 0.001 and p less than 0.001, respectively). Though the levels decreased during remission, they were still significantly above the mean level in the controls (p less than 0.05). The CIC level-disease status relationship was clearly evident in the serial studies on 19 CML patients who donated serum samples prior to treatment as well as during remission. Two-dimensional electrophoretic analysis of the CIC samples revealed the presence of a polypeptide (mol. wt approx. 37,000 and pI approx. 5.8) in 10 out of 18 CIC samples from the untreated CML patients. Such a moiety was not detected in six CIC samples from normal subjects. The association of this polypeptide with CML gains support from the observation that in 5 CML patients, this moiety was present in the CIC samples obtained prior to treatment but absent in the samples subsequently obtained during remission.

We here describe the cloning of an additional gene, called alpha 2-1.8, which is similar to the previously cloned human alpha 2-adrenergic receptor located on chromosome 4. The alpha 2-1.8 gene was identified by using the polymerase chain reaction with primers specific for sequences in transmembrane regions 2 and 5 of the previously isolated human alpha 2-C4 and alpha 2-C10 adrenoceptor genes, which are localized on chromosomes 4 and 10, respectively. The new gene was identified by amplifying the 1.8 kb size fractionated region of PstI restriction cut human genomic DNA. The previously cloned alpha 2-C10 and alpha 2-C4 genes were recovered at their expected locations, 0.96 and 5.9 kb, respectively. We have identified 387 bases of the new alpha 2-1.8 gene, and its sequence is identical to the previously described alpha 2-C4 gene, but it is distinct from the alpha 2-C10 and alpha 2-C2 genes. Our results demonstrate that the alpha 2-C4 adrenergic receptor exists in more than one copy in the human genome.