Partial purification and characterization of rhinoceros gonadotropins, growth hormone, and prolactin: comparison with the horse and sheep

The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purified fractions of LH, FSH, growth hormone (GH), and prolactin (PRL). LH was readily measured by a rat Leydig cell assay (0.1-1% x equine LH) and an RIA using a monoclonal antibody to bovine LH (6-11% x equine LH). FSH activity detected in the LH by either an FSH RIA or a calf testis radioreceptor assay (RRA) was extremely low. No FSH activity could be detected in the White Rhinoceros pituitary "FSH" fraction, but was readily detected in the Black Rhinoceros fraction (RIA: 0.2% x equine FSH: RRA: 0.8% x equine FSH). The presence of GH and PRL was determined by SDS-PAGE and Western blots. Results showed a single immunoreactive GH band and multiple immunoreactive PRL bands. Adsorption with Concanavalin A-Sepharose indicated that some of the PRL bands are glycosylated.     

Biological and immunoactive substances resembling chorionic gonadotropin are present in full-term horse and zebra placentas.

This study describes the presence of immunoactive and bioactive eCG-like material in full-term placentas of both domestic horses and zebras. Term placental extracts were immunoreactive in an LH monoclonal antibody RIA, and methods successfully used previously for the purification of eCG and eLH were employed to further concentrate the immunoreactive materials to the point where additional characterization studies could be performed. Sufficient equine material was obtained to perform a final fractionation on a concanavalin A Sepharose column yielding an unadsorbed fraction, e17A, and an adsorbed fraction, e17B. There was insufficient zebra material, z5D, for this step. HPLC gel filtration coupled with LH immunoassays of the column eluates showed all the final placental fractions to be highly heterogeneous, but a discrete peak of immunoactivity was found in one of the two equine fractions (e17B) and in the zebra fraction (z5D). The HPLC gel filtration elution volumes for e17B and z5D suggest that they have a smaller molecular size than either eCG or eLH but almost the same size as ovine LH. Both e17B and z5D were bioactive in the rat Leydig cell assay for LH but low in potency compared to eCG or eLH; e17A was inactive at very high doses (5 micrograms). This latter fraction, however, cross-reacted in an eCG alpha RIA to a much greater extent (6 times) than e17B, suggesting that it may be an incompletely formed or degraded alpha subunit. RIAs for LH, eCG, and eCG beta suggest that epitopes distinctive for these molecules are also present or similar to those in the term placental materials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zebra chorionic gonadotropin: partial purification and characterization

Six samples of pregnant zebra (z) serum from the first and second trimesters of pregnancy were analyzed by RIA and shown to have chorionic gonadotropin levels comparable to that of the mare (0.9-5.3 micrograms/ml); first trimester levels in most cases were higher than second trimester levels. A pool of the sera (10 ml) was fractionated by methods previously employed for the purification of equine (e) and donkey (d) chorionic gonadotropin to achieve a concentration of the zebra chorionic gonadotropin (zCG). A yield of 1.0 mg of glycoprotein was obtained. HPLC analysis of the material indicated the content of zCG to be about 7%. Its molecular size as judged by Ve/Vo values is smaller than eCG, greater than ovine LH, and about the same as equine LH. The zCG was tested in RIAs for LH and eCG, radioreceptor assays (RRA) for LH and FSH, and the rat testis Leydig cell assay for LH. Comparisons were made with equine and donkey chorionic gonadotropin, and equine and zebra LH. The results, preliminary because the preparation is not of high purity, showed that zCG is bioactive as an LH; immunologically similar to eCG, eLH, dCG, and zLH; and competes in RRAs for LH but not FSH receptors. It differs, therefore, from eCG and eLH--which have high levels of intrinsic FSH activity, and is more like dCG, dLH, and zLH--all of which have minimal if any FSH activity.     

Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.     

Detection of ruminal bacteria that degrade toxic dihydroxypyridine compounds produced from mimosine.

Leucaena leucocephala, a tropical leguminous shrub, contains a toxic amino acid, mimosine. Successful utilization of leucaena as a ruminant forage depends on colonization of the rumen by bacteria that degrade dihydroxypyridines (DHP), which are toxic intermediates in the metabolism of mimosine. Populations in the rumina of animals in some parts of the world, however, do not include bacteria that are able to carry out this degradation. We thus describe tests for the presence of DHP degraders in ruminal populations that are based on degradation (loss) of DHP compounds from culture media. Results obtained with the tests indicate that DHP degraders were not part of microbial populations in the rumina of cattle, sheep, and goats in Iowa, while most rumen samples examined from animals from the Virgin Islands and Haiti contained DHP degraders. These results confirm and extend the findings of others about geographic limits to the distribution of these important ruminal bacteria.     

Self-monitoring of blood glucose in diabetic pregnancy.

Admission to hospital is usually recommended to achieve the best possible diabetic control during pregnancy. We have used blood glucose monitoring at home to find out if patients can achieve equally good control outside hospital. Twenty-five consecutive diabetic patients were studied, of whom 20 had taken insulin before pregnancy. Six of their 14 previous pregnancies had ended in perinatal death. The 25 women performed 4247 blood glucose measurements during their pregnancies. Overall the mean blood glucose concentration was 7.1 mmol/l (128 mg/100 ml); before meals the mean was 6.5 mmol/l (117 mg/100 ml). Mean concentrations were lower in the third trimester, but at no stage was control in hospital significantly better than at home. The mean hospital stay before delivery was 22 days, and all patients had live babies. Monitoring blood glucose concentrations at home produces greater understanding and motivation among patients, improves control early in pregnancy, and shortens time spent in hospital.     

Increased brain myo-inositol 1-phosphate in lithium-treated rats

Lithium was found to produce a marked elevation in the levels of $\text{myo}$-inositol 1-phosphate in the cerebral cortex of treated rats. This effect was completely inhibited by atropine. A 40% reduction in the levels of $\text{myo}$-inositol 1-phosphate was observed when atropine was given alone.