Pegylated leptin antagonist with strong orexigenic activity in mice is not effective in chickens.

A chicken gene orthologous to human leptin receptor (LEPR) has been characterized and found to be active in leptin signaling in vitro in response to a variety of recombinant leptins and leptin-containing blood samples. However, the endogenous ligand of chicken LEPR (cLEPR) - the putative chicken leptin - has been reported by us and others to be undetectable at the DNA, mRNA, protein and activity levels. These reports have raised questions as to cLEPR's role. Here we analyzed the effects of a pegylated superactive mouse leptin antagonist (PEG-SMLA) in chicken. We showed that the leptin antagonist efficiently and specifically blocks leptin signaling through the cLEPR in vitro. The effect of the leptin antagonist was then studied in vivo by daily administration of 10 mg kg(-1) for 10 consecutive days to white leghorn female chickens (Gallus gallus) at the age of 2 weeks. Despites the efficient attenuation of the cLEPR in vitro, no effect was observed on body mass, feed intake, feed efficiency or fat accumulation in the treated birds. Because similar treatment in rodents leads to a highly pronounced increase in appetite and body mass that are observed from the first day of treatment, it is concluded that the cLEPR is not implicated in the control of appetite or adipose homeostasis in chickens.

Serum leptin activity in obese and lean patients.

Blood levels of the satiety hormone leptin are directly correlated to fat stores in obese and lean people. Therefore, leptin resistance is the logical explanation for the phenomenon of common obesity. However, the important question of whether or not the intrinsic leptin activity could differ between obese and lean people has not been examined before. In the present study, serum leptin activity was measured by an in vitro assay of leptin signaling in a modified culture of HEK-293 cells. The system is based on activation of a luciferase reporter gene through a leptin receptor-dependent activation of the signal transducer and activator of transcription (STAT3). Serum samples from 20 obese and 20 non-obese individuals with leptin levels ranging from 3 to 75 ng/ml, as determined by radioimmunoassay (RIA), were used. A high correlation was observed for each serum sample between leptin RIA values and leptin activity in the bioassay. The results indicate that obesity in the 20 obese patients among the 40 individuals examined cannot be accounted for by alterations in leptin activity in our assay. The assay system provides a tool to screen for possible rare cases exhibiting alteration in leptin activity either due to a change in leptin itself or through interaction with other serum factors.

Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

Carotid endarterectomy: creating the pathway to 1-day stay.

Use of a collaborative team approach to design a care pathway and standing orders for carotid endarterectomy patients achieved the project's goals. Variation, LOS, and resource consumption were decreased while quality of care and patients' satisfaction levels were maintained. Education of patients, patients' families, and staff members increased. Coordination between caregivers increased. The consistent concurrent database provided a feedback loop for continued change and for setting the target. Essential to the success was sponsorship from key leadership via the hospital's steering committee. The diverse membership of key associates on the CQI team helped to create an excellent revised carotid endarterectomy process and ensured full implementation. This membership of the CQI team was essential to comprehensive education and implementation. The step-by-step implementation kept the project moving forward. Creating a care pathway and changing practice require collaboration between nurses, doctors, and administrators. Creativity and systematic, thorough steps are what move a practice change from idea to inception.

SVMPA, a mutant of sindbis virus resistant to mycophenolic acid and ribavirin, shows an increased sensitivity to chick interferon.

SVMPA is a mutant of Sindbis virus, selected for its ability to replicate in mycophenolic acid (MPA)-treated mosquito cells. SVMPA has another phenotype: although able to replicate normally in primary cultures of chick embryo fibroblasts (CEF), its replication is restricted in secondary cultures prepared from aged primary CEF cultures. The mutations responsible for these phenotypes mapped to the region of the viral genome that codes for nsP1. We report here that SVMPA has yet another phenotype. Relative to our standard Sindbis virus (SVSTD) from which it was derived, SVMPA shows an increased sensitivity to chick interferon, both crude interferon prepared from virus-infected cells and recombinant interferon. Characterization of viral mutants obtained after site-directed mutagenesis indicated that the same mutations responsible for the host restriction of SVMPA in secondary cultures of CEF were also responsible for its increased sensitivity to chick interferon.

Calcium sensitivity and the effect of the calcium sensitizing drug pimobendan in the alcoholic isolated rat atrium.

We compared the effect of inotropic interventions (isoproterenol and pimobendan) and the relation between Ca2+ and isometric twitch force in atrial muscle from control rats and rats that had consumed alcohol for 2 months. At extracellular Ca2+ concentrations of 1-4 mM, alcohol atria developed less force than the controls. The median effective concentration (EC50) for extracellular Ca2+ was 3.2 +/- 0.01 mM for the alcohol group and 2.8 +/- 0.001 mM for the control group, whereas at maximal Ca2+, developed force was the same in both groups. To test whether the myofilament response to Ca2+ is altered with chronic alcohol consumption, we measured the relation between Ca2+ and force of atrial fiber bundle preparations extracted with Triton X-100. The Ca2+-force relation of alcohol atria (EC50 = 2.4 +/- 0.001 microM) was significantly shifted to the right of that of the control atria (EC50 = 1.94 +/- 0.001 microM Ca2+). Compared with controls, the alcohol atria demonstrated a significant depression in the inotropic effect of the beta-adrenergic agonist isoproterenol over a broad concentration range (10(-9)-10(-6) M). We also tested the effect of pimobendan, an inotropic agent with both phosphodiesterase-inhibiting and myofilament Ca2+-sensitizing actions. Developed force at concentrations of pimobendan <75 microM was similar between groups. However, at concentrations of pimobendan >75 microM, the developed force in alcohol atria was significantly less than control. Our results indicate that 2 months of alcohol consumption is associated with decreases in myofilament Ca2+ sensitivity and altered responsiveness to different inotropic agents.

A rapid, quantitative functional assay for measuring leptin.

At present, leptin is quantitated using immuno-assays that measure leptin mass. Leptin biological activity is determined using protocols that measure feed consumption and weight reduction. These in vivo protocols are semi-quantitative and require large quantities of leptin. We describe a rapid, sensitive and quantitative in vitro assay for leptin using HEK-293 cells stably co-transfected with the leptin receptor Ob-Rb isoform and a STAT-inducible promoter regulating the firefly luciferase cDNA. The assay, performed in a 96-well format, has an EC50 of 150 pM and is linear from 3 to 700 pM of leptin. We demonstrate that the assay is capable of measuring leptin in plasma samples. We demonstrate that bacterially-expressed, recombinant leptin and in vivo expressed leptin are equipotent. Furthermore, we demonstrate that a leptin-derived peptide, leptin fragment 22-56, previously shown to be capable of reducing feed intake following ICV injection does not act directly through the leptin receptor.

Cigarette smoking in a multiethnic population of youth: methods and baseline findings.

BACKGROUND: To expand upon recent research studies that have identified dramatic ethnic differences in adolescent cigarette smoking, this study was designed to characterize smoking among a multiethnic population of adolescents and to identify significant factors that may protect against smoking initiation. METHODS: During the first 2 years, this mixed cross-sectional, longitudinal study recruited and collected baseline data from a volunteer sample of 1,441 Houston-area public school students in the 5th, 8th, or 12th grade. A wide range of new and established predictors of smoking behavior was assessed, and their associations with ever smoking and susceptibility to smoking were assessed within ethnicity (white, N = 537; African-American, N = 454; and Hispanic, N = 297). RESULTS: Consistent with previous studies, white students smoked in substantially higher proportions than African-American students, with Hispanic adolescents in-between. Simultaneously adjusting for other variables, the odds of ever smoking (OR = 0.47, P < 0.01) and susceptibility to smoking (OR = 0.64, P < 0.01) were significantly lower among African-American adolescents when compared with whites; odds ratios for Hispanics and whites did not differ. Across all three ethnicities, the most important predictor of both ever smoking and susceptibility to smoking was the smoking status of the three best friends. Several ethnicity-specific variables also were identified. CONCLUSIONS: In concordance with previous investigations, cigarette smoking prevalence differs by ethnicity, and the factors associated with ever smoking and susceptibility to smoking differ among white, African-American, and Hispanic adolescents. The results of this study may be used to develop theory-based, culturally appropriate smoking intervention programs for adolescents.

Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse.

We have compared the efficacy of daily injection of recombinant leptin protein (rh-leptin) with adenovirus-mediated delivery of the murine or human leptin gene (Ad-leptin) for treatment of obesity in the obese (ob/ob) mouse model. We demonstrate an improved correction profile for obesity and associated surrogate markers using the adenovirus delivery method. Rate of weight loss and percentage satiety were significantly greater in the mice treated with Adleptin. These findings were associated with lower peak serum leptin levels with Ad-leptin (22.9 +/- 2.6 ng/ml for the human gene, and 48.9 +/- 11.5 ng/ml for the murine gene) compared to rh-leptin (385.2 +/- 36.0 ng/ml). (Values are given as mean +/- standard error of the mean.) Importantly rh-leptin and ex vivo-expressed Ad-leptin were equivalently active in a functional cell-based assay. The primary difference in the two therapeutic approaches is the continuous chronic secretion of leptin mediated by gene delivery, versus the intermittent bolus delivery and rapid clearance of the daily injection of rh-leptin protein. Thus, in vivo findings suggest that leptin effects are better achieved at lower steady-state levels, a pharmacological feature attained here by gene therapy. These findings may have implications for the potential use of leptin in the treatment of obesity.

Production of leptin in Escherichia coli: a comparison of methods.

A procedure is described for gram-scale refolding of Escherichia coli-derived human leptin inclusion bodies. Refolding was achieved by gradually reducing denaturant using a diafiltration method. Refolded leptin is characterized by in vivo modulation of food intake, reduction in body weight, and lowering of insulin and glucose levels in ob/ob mice. In addition, refolded leptin is characterized by radioimmunoassay (RIA) and activation of the leptin receptor in a cell-based assay. For comparison we also refolded leptin by a simple dilution method and produced periplasmic derived leptin, which did not require ex vivo folding. Leptin produced by these three methods and leptin obtained from commercial sources were compared using the RIA and the cell-based assay and appeared to be of comparable quality and potency.

Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Functional STAT 1 and 3 signaling by the leptin receptor (OB-R); reduced expression of the rat fatty leptin receptor in transfected cells.

The leptin receptor (OB-R) bears homology to members of the class I cytokine receptor family. We demonstrate that leptin binding to OB-R stimulates formation of STAT-1 and STAT-3 complexes, thereby defining transcriptional motifs for genes that are under leptin control. Transfected fa OB-R bound leptin with equal affinity to that of wild type OB-R. fa OB-R abundance was about 7 fold reduced compared to control cells. Surprisingly, the low level of fa OB-R is fully capable of activating the STAT signal transduction pathway. We discuss plausible explanations for the obese phenotype in Zucker fatty rats.

A receptor in pituitary and hypothalamus that functions in growth hormone release.

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

In ovo transfection of chicken embryos using cationic liposomes.

It is reported that cationic liposomes are capable of transfecting embryos in unincubated fertile chicken eggs and that the cationic liposome, TransfectAce, has superior properties to Lipofectin. In order to determine the duration of expression of genes introduced in this way, embryos were transfected with an expression vector encoding the firefly luciferase cDNA under the control of the Rous sarcoma virus long terminal repeat (LTR). Luciferase activity could be observed consistently in day 3 embryos and activity was detectable up to day 8 of incubation. The relative expression of luciferase under the control of different viral promoters was compared in transfected chicken embryo fibroblasts and day 3 embryos. The cytomegalovirus immediate early promoter and the SV40 early promoter directed the highest amount of expression in fibroblasts while the Rous sarcoma virus LTR caused the highest amount of expression in embryos. Chicken embryo fibroblasts were transfected with the luciferase vector in order to examine duration of reporter gene expression in vitro. Luciferase expression was decreased exponentially over a 24-day period after which point luciferase activity could no longer be detected. These data suggest that stable integration of transfected DNA using liposomes is a rare event. Nevertheless, liposome-mediated transfection of embryos is suitable for the examination of promoter activity in vivo and may be a useful method to transfect genes to study embryonic development.

Mutations in the nsP1 coding sequence of Sindbis virus which restrict viral replication in secondary cultures of chick embryo fibroblasts prepared from aged primary cultures.

SVMPA, a mutant of Sindbis virus (SV), which is able to replicate in Aedes albopictus cells treated with mycophenolic acid (MPA) or ribavirin, also has a host range phenotype. This phenotype is most clearly demonstrated by means of efficiency of plaquing (EOP) assays on secondary chick embryo fibroblasts (CEF) prepared from aged primary CEF. For example, in one experiment in which standard SV (SVSTD) had a relative EOP (EOP on primary CEF divided by EOP on secondary CEF) of 1.6 the corresponding value for SVMPA was 2340. The host restriction of SVMPA was also seen with similarly prepared secondary cultures of duck embryo fibroblasts, but not with established lines of quail cells. The finding that the accumulation of viral RNA was much lower in SVMPA-infected secondary CEF than in SVSTD-infected CEF indicated that the replication of SVMPA in these cultures was blocked at an early step. Revertants of SVMPA were isolated which were no longer host-restricted but had retained their resistance to MPA. Of the three mutations [nucleotide (nt) 120, 127, and 963] in the nsP1 coding sequence of SVMPA, which lead to amino acid changes, these revertants had retained the nt 127 and nt 963 mutations but had lost the nt 120 mutation. This, along with earlier findings, indicated that only the nt 127 and nt 963 mutations are required for resistance to MPA. This result also associated the nt 120 mutation with the host restriction phenotype. In other experiments derivatives of pToto1101 (a plasmid from which infectious Sindbis virus RNA can be transcribed) were constructed by site-directed mutagenesis and used to test the effect of specific mutations on the viral phenotype. Although we were unable to obtain viable virus with the nt 120 mutation alone, virus with the nt 120 and nt 127 mutations was viable and host-restricted. We suggest that the nt 120 mutation by itself is lethal and that the nt 127 mutation suppresses the lethal effect of the nt 120 mutation. The SVMPA mutation at nt 120, which is associated with the host range phenotype, changes Gln21 of nsP1 to Lys. When a more conservative change was engineered, i.e., to Asn, the virus was not host-restricted. Although the reason for the restriction of SVMPA replication in secondary CEF is not known, some possible explanations are discussed.

Avian retroviral expression of luciferase.

Biologically active replication-competent (subgroups A, B, and C) and replication-defective Rous sarcoma virus-derived vectors containing the cDNA encoding firefly luciferase as a reporter gene were constructed. In these retroviral vectors, luciferase is expressed from a spliced subgenomic mRNA. A biologically active replication-defective UR2 virus-derived vector expressing the reporter gene as a gag-luciferase fusion protein from an unspliced genomic mRNA was also constructed. The luciferase reporter gene was used because it lacks homology with chicken genomic sequences and because a rapid and sensitive direct enzymatic assay is available to monitor luciferase expression in retrovirus-infected cells. The levels of luciferase expression in luciferase recombinant retrovirus-infected chicken embryo fibroblasts are greater than 10(3) higher than that detected in uninfected cells or in cells infected with retroviral vectors carrying other genes. Endpoint dilution titration experiments demonstrated that one infected cell can be detected in a background of 10(3) uninfected cells. The vectors are stable in tissue culture and high level expression of the unselected luciferase reporter gene is maintained. The vectors were used to express luciferase in chicken embryos, demonstrating the potential utility of luciferase as a reporter in vivo.

Broiler chicks fed low-calcium diets. 2. Increased sensitivity to copper toxicity.

Young broiler chicks were more sensitive to copper toxicity when they were fed diets deficient or marginal in calcium content. Growth rate was depressed and liver copper concentration was increased under these conditions. Chicks fed a casein-gelatin diet were more sensitive to copper toxicity than those fed a corn-soybean meal diet. Addition of phytic acid to the casein-gelatin basal diet enhanced copper toxicity as evidenced by effects on growth rate and liver copper content. Measurements of intestinal and biliary copper content suggested that the influence of calcium on copper toxicity was mediated via intestinal absorption rather than through influences on copper excretion.

Diminished hepatic growth hormone receptor binding in sex-linked dwarf broiler and leghorn chickens.

Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

Purification and physiochemical properties of a recombinant bovine growth hormone produced by cultured murine fibroblasts.

Mouse fibroblast cell lines which secrete bovine (b) GH have been generated. This was accomplished by cotransforming mouse L cells (thymidine kinase-negative [TK-] and adenine phosphoribosyl transferase-negative [APRT-]) with DNA molecules encoding the Rous sarcoma virus-long-terminal repeat and bGH genes along with the herpes viral TK gene and the hamster APRT gene. One stable cell line, Pd lambda-pbGH 4-13, was found to secrete approximately 75 micrograms bGH per 24 h/5.0 X 10(6) cells. Media from this cell line were collected for purification of recombinant bGH (rbGH). Purification involved (NH4)2SO4 fractionation, ion-exchange chromatography, and gel filtration on Sephacryl S-200. The rbGH was characterized by bioassay, RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis. Results of these analyses were compared with those obtained with a highly purified pituitary bGH. In the rat tibia bioassay, rbGH was found to be as potent as pituitary bGH. Results from the RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis and Western blot analysis also suggested that the rbGH was similar to that of pituitary origin. Amino acid composition, partial (amino-terminal) sequence, and tryptic peptide maps were also found to be similar between the rbGH and pituitary bGH preparations. The amino terminus of the rbGH showed similar heterogeneity to that of the bGH of pituitary origin. We conclude that rbGH which was synthesized, processed, and secreted from transformed mouse fibroblasts possessed almost exactly the same physiochemical properties as pituitary bGH.