Multiple central alpha 2 adrenoceptors of avian and mammalian species.

Although equilibrium binding experiments indicated that calf cerebral membranes contained two classes of clonidine receptors and that chicken cerebral membranes might have contained only one, experiments investigating the kinetics of binding and the effects of GppNHp clearly indicated that the cerebral membranes of both species contained two subtypes of receptor, with the avian high affinity receptor having been present at too low a density to be readily detected in equilibrium binding studies. For both species 10 microM GppNHp sharply reduced or eliminated both the high affinity binding site and the slow steps of association and dissociation without changing the low affinity site and its related rapid association and dissociation steps. The high affinity sites from both species had similar specificities since the relative affinities of the avian binding site for a series of clonidine analogues closely reflected the relative affinities of the calf binding site. The properties of the chicken and calf alpha 2 subtypes resembled those reported for rat brain.

Mass spectrometric analysis of the fatty acids and nonsaponifiable lipids of Eimeria tenella oocysts.

The fatty acids and nonsaponifiable lipids of Eimeria tenella oocysts were analyzed by gas liquid chromatography and combined gas liquid chromatography-mass spectrometry. The fatty acids detected were identified as C14:0, C16:0, C16:1, C18:0, C18:1, and C18:2. Though the wt of the fatty acid fraction decreased during sporulation from 91 mug per 10(6) oocysts to 47 mug per 10(6) oocysts, the relative amounts of these fatty acids did not change appreciably. The nonsaponifiable lipids of E. tenella consisted of cholesterol and unbranched primary alcohols of 22, 24, 26, 28, 30, and 32 carbons. Mass fragmentography demonstrated that each species of alcohol consisted of saturated and monounsaturated derivatives. Trimethylsilyl ethers of fatty alcohols were found to offer several important advantages over free alcohols for mass spectrometric characterization. Before sporulation, most fatty alcohols were in the oocyst wall. During sporulation, the wt of the nonsaponifiable lipids increased from 16 mug per 10(6) oocysts to 44 mug per 10(6) oocysts due largely to synthesis of C24 and C26 alcohols. The newly synthesized fatty alcohols were not deposited in the oocyst wall.

Comparison of anticoccidial efficacy, resistance and tolerance of narasin, monensin and lasalocid in chicken battery trials.

The anticoccidial efficacy, host tolerance, and projected resistance development of the three polyether antibiotics, monensin, narasin, and lasalocid were compared. The efficacy of narasin against different coccidial strains was found to parallel that of monensin in as much as strains which were refractory to monensin were also refractory to narasin. In contrast, lasalocid easily controlled some strains which were not well controlled by either narasin or monensin and failed to control one strain readily controlled by these two antibiotics. In growing chicks, lasalocid at the projected use level of 75 p.p.m. and narasin at an efficacious level of 100 p.p.m. were both better tolerated than monensin at the recommended use level of 121 p.p.m. The frequency of mutants resistant to each of these polyether compounds was found to be less than 8.6 X 10(-9) per drug sensitive oocyst for one strain of Eimeria tenella. This corresponds to less than 0.036 and 0.148 as frequent as mutants of this strain resistant to glycarbylamide or to amquinate, respectively.

Battery efficacy studies with arprinocid against field strains of Coccidia.

Graded levels of 9-[2-chloro-6-fluorophenyl)methyl]-9H-purin-6-amine (arprinocid) ranging from 30 ppm to 90 ppm in the diet have been tested for anticoccidial efficacy in controlled battery experiments against 20 field isolates of coccidia. These isolates, representing five major species of Eimeria, were collected from various geographic locations and all but one were found to be resistant to one or more of the currently marketed products. Arprinocid prevented mortality, increased weight gains, and reduced or eliminated intestinal lesions and oocyst production. A level of 60 ppm arprinocid provided adequate control of all isolates and no evidence of cross resistance was observed between arprinocid and the marketed products tested. On the basis of these studies, it is concluded that arprinocid at levels of 60 ppm to 70 ppm is effective in controlling coccidiosis in broilers, even that caused by isolates refractory to many of the marketed products.

Effects of gonadal steroids and adrenergic agonists on avian growth and feed efficiency.

The effects of gonadal steroids and adrenergic agonists in growing poultry are reviewed. Estrogens, used commercially to caponize, improve muscle palatability and weight gain but are hyperlipidemic and adversely affect feed efficiency. Their ability to caponize results from the fact that negative feedback regulation of gonadotropin secretion is mediated by estrogen synthesized within the male's central nervous system. Androgens are strongly anabolic in avian species but are deleterious to growing bone. Thus, androgens depress growth and efficiency when given before epiphyseal closure but stimulate gain and improve efficiency when given afterwards. The beta-adrenergic agonist clenbuterol improves the growth and efficiency of broiler chickens. The effects of beta-agonists on muscle, adipogenic tissue, and the endocrine system are presented as possible mechanisms for these actions.

Regulation of nonspecific acid phosphatase in Salmonella: phoN and phoP genes.

Mutations in Salmonella typhimurium strains lacking nonspecific acid phosphatase mapped in two unlinked loci. One of these, phoP, was cotransducible by phage P22 with purB, whereas the second, phoN, was cotransducible by phage P1 with purA. Mutants with temperature-sensitive nonspecific acid phosphatase activity (measured in whole cells) were also isolated. A phoN mutant with thermolabile whole-cell activity was isolated directly from wild-type LT-2. Several other mutants with temperature-sensitive enzyme activity were also isolated as revertants of phoN mutants. These data suggest that phoN might be a structural locus for nonspecific acid phosphatase. The observation that a mutation resulting in high level of nonspecific acid phosphatase mapped in phoP suggests a possible regulatory role for this locus.

Isolation of amylopectin granules and identification of amylopectin phosphorylase in the oocysts of Eimeria tenella.

Amylopectin granules were purified from Eimeria tenella oocysts following digestion with sodium dodecyl sulfate and pronase. The oval granules had a uniform size of 0.5 X 0.7 mum, and consisted of only glucose polymers. alpha-Amylase treatment yielded 235 nmoles of maltose from the granules from 10(6) unsporulated oocysts and 93 nmoles maltose from those from 10(6) sporulated oocysts. Amylopectin phosphorylase activity was detected in the cytoplasm of unsporulated oocysts of E. tenella. It had a specific activity of 13 U/mg protein in crude extracts, and a pH optimum of 6.0. The Km values determined were 9.1 mM for glucose-1-phosphate and 5.6 mM for glucose end groups in potato amylopectin. Enzyme activity declined at a linear rate during sporulation, sporulated oocysts containing less than 8% of the activity of unsporulated oocysts. No amylase-type activity was found in the parasite.

Purification and characterization of avian dopamine beta-hydroxylase.

Dopamine beta-hydroxylase (EC 1.14.17.1) has been purified from the chromaffin granules of avian adrenals. The enzyme has a molecular mass of approximately 320K daltons and consists of four apparently identical subunits joined in pairs by disulfide bonds. Analysis of the products formed from dopamine tritiated in the beta position indicated that 1.72 times as much tritium was retained in norepinephrine as was released as water. Ferrocyanide could serve as a reductant, but ascorbate at equal concentrations afforded higher rates. The enzyme had a pH optimum of 5-6 and was activated by either fumarate or acetate, with fumarate being far more effective. Kinetic experiments varying the concentrations of the substrates ascorbate and dopamine and those of the products dehydroascorbate and norepinephrine suggested that the mechanism was un-uni bi-uni ping pong. By this mechanism, the enzyme released dehydroascorbate after being irreversibly reduced by ascorbate and then sequentially bound oxygen and dopamine and released the product norepinephrine. The enzyme was inhibited by high but probably physiological concentrations of the substrate ascorbate and was activated by low concentrations of the product dehydroascorbate. Ascorbate inhibition was noncompetitive with dopamine, and dehydroascorbate activation was due to an increase in the enzyme's affinity for ascorbate with little or no change in its Vmax. Substrate inhibition by ascorbate and product activation by dehydroascorbate might together ensure that the rate of norepinephrine synthesis in vivo remains relatively unaffected by changes in the ratio of ascorbate to dehydroascorbate within chromaffin granules.

Antifertility effects of clonidine in laying hens.

Clonidine was anovulatory and markedly antigonadal in laying hens when infused for 1 week from minipump implants at daily rates of 1.08 mg per hen or greater. The ovaries of hens treated with clonidine responded to FSH injections which suggests that the antigonadal effect of clonidine resulted from a reduction in the output of gonadotropin by the pituitary. These data suggest that alpha 2 receptors may be important in regulating avian fertility.

Effect of tamoxifen on H-Y antigen expression and gonadal development in chicken embryos.

Avian species follow the ZW/ZZ system of sex determination, which the female is heterogametic and expresses H-Y (or, more appropriately, 'H-W') antigen. We present the results of an investigation into the effects of the antiestrogen, tamoxifen, on gonadal differentiation and H-Y antigen expression in chickens. When given at doses of 0.25-2 mg per egg immediately before incubation, tamoxifen blocked regression of the right gonad in a significant number of 14-day-old female embryos. The nonregressed right gonad had a testis-like external appearance and, in some cases, contained what appeared to be spermatogenic tubules. Tamoxifen had no histologically detectable effect on the differentiation of the left ovary or the testes. In spite of tamoxifen's histological effects on right female gonads, it did not masculinize the steroidogenic capabilities of these gonads. Whether obtained from drug- or vehicle-treated embryos, the left and right female gonads always contained appreciable amounts of estrogen. In contrast, testes obtained from either drug- or vehicle-treated embryos did not contain detectable amounts of estrogen. Tamoxifen reduced the H-Y antigen levels in female liver and gonads. In both left and right female gonads, the reduction was to male levels. In female livers, tamoxifen reduced H-Y antigen to levels intermediate between those of normal males and females. Thus, the expression of H-Y antigen in both gonadal and nongonadal tissue is estrogen dependent, but the dependency appears to be more stringent for gonadal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The antifertility and antiadrenergic actions of thiocarbamate fungicides in laying hens.

The effects of the thiocarbamate fungicides, thiram, ziram, ferbam, maneb, and zineb, on norepinephrine synthesis by laying hens were investigated. Inhibition experiments with dopamine beta-hydroxylase purified from chicken adrenals indicated that thiram, ziram, and ferbam are potent competitive inhibitors with the substrate the substrate ascorbate. Maneb and zineb were without effect at comparable concentrations. Experiments investigating the interaction of thiram, ziram, and ferbam with cupric ions suggested that these compounds probably inhibit the enzyme by complexing the fully oxidized copper at its active site. Maneb and zineb also complexed cupric ions in solution and thus their failure to inhibit is not due to their inability to complex copper. When tested in vivo, thiram, ziram, and ferbam at po doses of 2.5 mg/kg or greater significantly reduced the conversion of radioactive dopa, given systemically, to brain norepinephrine. Since they did not affect the uptake of radioactive dopa by the brain or its subsequent decarboxylation within the brain to yield dopamine, these three compounds inhibit cerebral dopamine beta-hydroxylase in vivo. In contrast maneb and zineb at a po dose of 20 mg/kg had no significant effect on brain norepinephrine synthesis. Previously published results (Weppelman et al., Biol. Reprod. 23, 40-46, 1980) demonstrated that thiram, ziram, and ferbam (but not maneb or zineb) have antifertility action in laying hens. The correlation between this action and inhibition of dopamine beta-hydroxylase suggests that the antifertility effects of thiram, ziram, and ferbam might result from their antiadrenergic action. The observation that all doses of thiram in the diet which caused significant antigonadal action when fed to laying hens for 1 week also significantly decreased central and peripheral stores of norepinephrine supports this conclusion.

Purification and properties of chicken growth hormone and the development of a homologous radioimmunoassay.

Highly purified growth hormone (GH) has been isolated from pituitary glands of chicken (Gallus domesticus), and a specific homologous radioimmunoassay (RIA) has also been developed. The purified chicken GH was active in the rat tibia bioassay and it gave a dose-dependent response which paralleled that of the bovine GH standard. High pressure liquid chromatography revealed that the purified chicken GH was homogenous. Chicken GH had an Rf value of 0.2 in disc electrophoresis, and a MW of 26,000 from sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was estimated to be 7.6 by gel isoelectric focusing. The amino acid composition of chicken GH was found to be similar to that of mammalian GH, and the NH2-terminal amino acid was threonine. Partial sequencing (114 amino acids) of the chicken GH showed 79% homology with bovine GH. An antiserum was developed to the purified chicken GH in a rabbit, and it was used to develop a homologous RIA using 125I-labeled chicken GH as the ligand. The purified chicken GH was iodinated via the lactoperoxidase method to a specific activity of approximately 100 microCi/micrograms. Plasma from chickens, medium from incubation of pituitary glands, and homogenates of pituitary glands gave parallel dilution-response curves with the chicken GH standard. Mammalian GH, prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) showed no cross-reaction with the 125I-labeled chicken GH. Purified turkey GH showed parallel dose response with the chicken GH, but purified turkey PRL did not cross-react. Chicken FSH and LH also showed no inhibition of binding. The minimum detectable concentration of the assay was 0.93 ng/tube, and the intraassay and interassay coefficients of variation were 9 and 16%, respectively. The specific binding of 125I-labeled chicken GH to a microsomal fraction isolated from chicken liver was identified, and the specific binding was generally low (1-4%). Turkey PRL, and chicken LH and FSH showed no inhibition of the 125I-labeled chicken GH hepatic binding and the ontogeny of the hepatic GH receptor binding sites in male and female chickens was examined.