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The G protein-coupled receptor 83 (GPR83) was recently demonstrated in warm sensitive neurons (WSN) of the hypothalamic preoptic area (POA) that participate in temperature homeostasis. Thus, we investigated whether GPR83 may have a role in regulating core body temperature (CBT) by reducing its expression in the POA. Dissipation of energy in the form of heat is the primary mode of energy expenditure in mammals and can ultimately affect energy homeostasis. Thus, we also measured the level of important regulators of metabolism. Downregulation of GPR83 was obtained by lentiviral short-hairpin RNAs (shGPR83) vectors designed and selected for their ability to reduce GPR83 levels in vitro. Mice received POA injection of shGPR83 or non-silencing vectors and were monitored for CBT, motor activity, food intake body weight and circulating levels of IGF-1, insulin, leptin and adiponectin. Down-regulation of GPR83 in the POA resulted in a small (0.15°C) but significant reduction of CBT during the dark/active cycle of the day. Temperature reduction was followed by increased body weight gain independent of caloric intake. shGPR83 mice also had increased level of circulating adiponectin (31916±952 pg/mL vs. 23474±1507 pg/mL, P<.01) while no change was observed for insulin, IGF-1 or leptin. GPR83 may participate in central thermoregulation and the central control of circulating adiponectin. Further work is required to determine how GPR83 can affect POA WSN and what are the long term metabolic consequences of its down-regulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Adiponectin can act in the brain to increase energy expenditure and reduce body weight by mechanisms not entirely understood. We found that adiponectin type 1 and type 2 receptors (AdipoR1 and AdipoR2) are expressed in warm sensitive neurons of the hypothalamic preoptic area (POA) which play a critical role in the regulation of core body temperature (CBT) and energy balance. Thus, we tested the ability of adiponectin to influence CBT in wild-type mice and in mice deficient for AdipoR1 or AdipoR2. Local injection of adiponectin into the POA induced prolonged elevation of core body temperature and decreased respiratory exchange ratio (RER) indicating that increased energy expenditure is associated with increased oxidation of fat over carbohydrates. In AdipoR1 deficient mice, the ability of adiponectin to raise CBT was significantly blunted and its ability to decrease RER was completely lost. In AdipoR2 deficient mice, adiponectin had only diminished hyperthermic effects but reduced RER similarly to wild type mice. These results indicate that adiponectin can contribute to energy homeostasis by regulating CBT by direct actions on AdipoR1 and R2 in the POA., (Copyright © 2011 Elsevier B.V. All rights reserved.)

CC Chemokine ligand 22 (Ccl22) is a selective, high affinity ligand at the CC chemokine receptor 4 (Ccr4). We have identified cDNAs encoding both ligand and receptor of the Ccl22-Ccr4 pair in cDNA libraries of the anterior hypothalamus/pre-optic area (AH/POA) by PCR. The AH/POA is the key brain region where endogenous pyrogens have been shown to act on warm sensitive neurons to affect thermogenesis in brown adipose tissue (BAT) and other thermogenically responsive tissues. We show that functional Ccr4 receptors are present in the AH/POA neurons as injection of Ccl22 into the POA but not to other hypothalamic nuclei induces an increase in core body temperature as measured by radiotelemetry. Indomethacin (5 mg/kg s.c) pre-treatment markedly reduced the hyperthermia evoked by POA injection of Ccl22 (10 ng/0.5 ul) and thus suggests that this hyperthermia is mediated through cyclooxygenase activation and thus likely through the formation and action of the pyrogen prostaglandin E2. The temperature elevation involves a decrease in the respiratory exchange ratio and increased activation of the brown adipose tissue as demonstrated by ¹⁸F-FDG-PET imaging. We describe a novel role to the ligand Ccl22 and its receptor Ccr4 in the anterior hypothalamus in temperature regulation that depends on the synthesis of the endogenous pyrogen, prostaglandin E2., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We describe the design, synthesis, and structure-activity relationships of triazolobenzodiazepinone CCK1 receptor agonists. Analogs in this series demonstrate potent agonist activity as measured by in vitro and in vivo assays for CCK1 agonism. Our efforts resulted in the identification of compound 4a which significantly reduced food intake with minimal systemic exposure in rodents., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Peptidic glucagon antagonists have been shown to lower blood glucose levels in diabetic models (1-3), but attempts to identify small molecular weight glucagon receptor-binding antagonists have met with little success. Skyrin, a fungal bisanthroquinone, exhibits functional glucagon antagonism by uncoupling the glucagon receptor from adenylate cyclase activation in rat liver membranes (1). We have examined the effects of skyrin on cells transfected with the human glucagon receptor and on isolated rat and human hepatocytes. The skyrin used was isolated from Talaromyces wortmanni American Type Culture Collection 10517. In rat hepatocytes, skyrin (30 micromol/l) inhibited glucagon-stimulated cAMP production (53%) and glucose output (IC50 56 micromol/l). There was no detectable effect on epinephrine or glucagon-like peptide 1 (GLP-1) stimulation of these parameters, which demonstrates skyrin's selective activity. Skyrin was also evaluated in primary cultures of human hepatocytes. Unlike cell lines, which are largely unresponsive to glucagon, primary human hepatocytes exhibited glucagon-dependent cAMP production for 14 days in culture (EC50 10 nmol/l). Skyrin (10 micromol/l) markedly reduced glucagon-stimulated cAMP production (55%) and glycogenolysis (27%) in human hepatocytes. The inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones. Skyrin is the first small molecular weight nonpeptidic agent demonstrated to interfere with the coupling of glucagon to adenylate cyclase independent of binding to the glucagon receptor. The data presented in this study indicate that functional uncoupling of the human glucagon receptor from cAMP production results in metabolic effects that could reduce hepatocyte glucose production and hence alleviate diabetic hyperglycemia.

Mouse lymphoma cells of the L5178Y TK + /-3.7.2C line were exposed to varying concentrations of the anti-schistosomal drug hycanthone methanesulfonate. The trifluorothymidine (TFT)-resistant cells fell into two classes based on colony size. Southern blot analyses were performed using NcoI-digested DNA from a number of large and small mutant colonies from each treatment group. Two different restriction fragment banding patterns were identified in these analyses, those colonies that contained the 6.4 kb NcoI restriction fragment and those that did not. A total of 471 mutant colonies were analyzed and 84.5% (398) of these colonies did not exhibit the 6.4 kb fragment. There did not appear to be a hycanthone methanesulfonate dose response effect in the number of colonies that did not contain the 6.4 kb fragment among the treated groups. In addition, 82% (154 out of 188) of spontaneous mutants did not contain the 6.4 kb fragment. The results imply that greater than 80% of all spontaneous mutations found in the mouse lymphoma assay regardless of colony size do not contain the 6.4 kb fragment and each may be considered to be a large scale mutation. In addition, greater than 80% of the hycanthone induced mutations in the mouse lymphoma assay do not contain the 6.4 kb fragment and thus may be considered to be a large scale mutation.

Inflammation, metastasis and ischemia are processes that require lymphocyte or leukocyte cell recognition and adherence to endothelial counter receptors such as ICAM-1. Mapping the sites of interaction of ICAM-1 with LFA-1, the receptor for ICAM-1 on lymphocytes, may lead to the design of novel inhibitors of inflammation or metastasis. To this end, recombinant soluble ICAM-1 cDNA was engineered into the baculovirus expression system, which is capable of expressing large amounts of proteins. These constructs were designed to contain a protein leader sequence so that the transfected insect cells would secrete the recombinant polypeptide into the culture media for ease of isolation. We engineered four constructs of ICAM-1 into the baculovirus system and obtained relatively high expression of two soluble forms of ICAM-1, a two domain and a five domain form. These truncated proteins were isolated and shown to promote adherence of HL-60 cells and Molt-4 cells. These recombinant soluble proteins also inhibited cell adherence to purified intact ICAM-1 isolated from K562 cells.

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.

Many metals have been shown to alter the function of a wide range of enzyme systems, including those involved in DNA repair and replication. To assess the impact in vivo of such metal actions a "Microtitre" fluctuation assay was used to examine the ability of Ni(II) to act as a comutagen with simple alkylating agents. In E. coli, Ni(II) chloride potentiated the mutagenicity of methyl methanesulfonate (MMS) in polymerase-proficient strains (WP2+ and WP2-), but not in polA- strains (WP6 and WP67) or in lexA- (CM561) or recA- (CM571) strains. The absence of UV excision repair (WP2- and WP67) had little, if any, effect. An extended lag phase was seen at 2-4 h in the polA- strains following treatment with Ni(II) chloride and MMS, but normal growth resumed thereafter. Results suggested that mutations induced by MMS were fixed during log phase growth and that more than 2 h of exposure were necessary for potentiation by Ni(II) to be observed. Thus, the extended lag phase probably cannot explain the lack of potentiation. RecA-dependence of the comutagenic effect was corroborated with S. typhimurium TA1535 and TA100. Only in the pKM101 containing strain, TA100, was potentiation of ethyl methanesulfonate (EMS) and MMS by Ni(II) chloride evident. The mucAB genes carried on pKM101 increase the sensitivity of TA100 to a variety of mutagens, providing there is a functional recA gene product. Taken together, the data suggest that Ni(II) acts indirectly, as a comutagen, in bacterial systems, possibly affecting processes involving recA- and/or polA-dependent function(s).

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.

A collection of 81 his-3 mutants of Neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. In these studies, the linearity of the complementation map of the his-3 cistron (Webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. In the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment with the chemical mutagens N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino) acridine dihydrochloride, nitrous acid or hydroxylamine. The frequency of revertible mutants among the non-polarized complementing mutants was 96% (45/47), and 79% (15/19) for the polarized complementing and 79% (11/14) for the non-complementing mutants. The results of these classical genetic assays for allelic complementation and specific revertibility suggest a correlation between complementation pattern and presumptive genetic alterations at the molecular level among his-3 mutants similar to that found with ad-3B mutants induced by nitrous acid (Malling and de Serres, 1967), ethyl methanesulfonate (Malling and de Serres, 1968), or ultraviolet (Kilbey et al., 1971).

Previous studies (Overton et al., Mutation Res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3B mutants. In the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. The restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-OR23-1A and were consistent with expectations based on previous data suggesting that they resulted from single base-pair alterations (Overton et al., Mutation Res., 1989). His-3 mutants with altered banding patterns were only found among those with polarized complementation patterns or noncomplementing mutants. One of the mutants with a polarized complementation pattern, 1-189-83, and another noncomplementing mutant, 1-189-85, are associated with genetic alterations proximal to the his-3 locus. In one other mutant, 1-226-565 (with a polarized complementation pattern), an insertion of approx. 2 kb has occurred in the proximal region of the his-3 locus. Two other mutants, 1-155-270 and 1-155-276 (both noncomplementing), contained a large insertion of approx. 12.8 kb in the proximal region of the his-3 locus.