Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in pulmonary Cu−Zn contents in superior mesenteric artery occlusion shock of rabbit

Contents of Cu and Zn of into-pulmonary blood (IPB), out-pulmonary blood (OPB), Lung tissue, and supernatant and macrophages of Lung Lavage were determined in superior mesenteric artery occlusion (SMAO) shock of rabbits. Zn of pulmonary tissue was 11.42 +/- 0.60 and 14.52 +/- 1.78 (micrograms/g wet wt) in SMAO shock and control groups, respectively. Content of Zn was found to be lower, Cu was not changed, and Cu/Zn ratio increased in lung tissue in SMAO shock. Contents of Cu and Zn in other samples were not changed. The results suggest that lower Zn in lung tissue related to acute lung injury.     

Sialic acid moiety is responsible for the charge heterogeneity and the biological potency of rat lutropin

To elucidate a possible role of sialic acid moiety in the electrical heterogeneity of rat pituitary lutropin, seven components separated were individually treated with neuraminidase. Some intermediates with isoelectric points corresponding to the native components were concomitantly seen at the serial stages of the enzyme treatment. All the treated components showed an isoelectric point of about 10.0 which was the same to the isoelectric point of one of the seven components. Desialylation of the components with less biological activity caused enhancement of the in vitro cyclic AMP producing- and testosterone producing-activities as well as the binding activity to the receptor. It is concluded that the number of sialic acid moiety in lutropin is responsible for the charge heterogeneity and the biological potency of the hormone.     

Transport of imino acids and non-α-amino acids across the brush-border membrane of the rabbit ileum

Summary The transport of -alanine and MeAIB and their effects as inhibitors of the transport of alanine, leucine and lysine across the brush-border membrane of the intact epithelium from the rabbit's distal ileum has been examined. Two separate transport systems have been characterized: 1) A sodium-dependent, -alanine-accepting system, which is a high-affinity transport system for -amino-monocarboxylic acids (neutral a.a.) and for cationic a.a., accepts non--amino acids as well as non--imino acids, is moderately stereospecific, and for which the affinity of a neutral a.a. is greatly reduced by N-methylation. 2) A sodium-dependent transport system for imino acids, which is inaccessible to cationic amino acids and non--amino acids but accepts cyclic, non--imino acids, is moderately stereospecific, and for which neutral a.a. have much lower affinities than their N-methylated derivatives. On the basis of the observations of this and the preceding paper five transport systems for amino acids are ascribed to the rabbit ileum. Some discrepancies between the present results and those obtained with brush-border membrane microvesicles from the rabbit small intestine are discussed.     

Effect of clomiphene citrate on the rabbit ovary

Summary The effect of clomiphene citrate on the rabbit ovary was studied in mature nulliparous rabbits pretreated with three consecutive doses ranging from 0.01–10.0 mg/kg per day. With increasing doses a trend of decrease in mean ovarian weight (mg/kg body weight) is observed 2 days after termination of treatment. Five days later a significant increase occurs, which then subsides again to control values on day 12 after termination of treatment. During this period, no matings or injections of luteinizing hormone were performed to trigger ovulation; consequently no ovulations are observed. Folliculogenesis appears as normally; number and morphology of follicles are within normal ranges. No endogenous, spontaneous gonadotropin surges are detected in blood serum up to the 7th day after termination of treatment (2 and 10 mg doses). The surface epithelium of the ovary resembles normal germinal epithelium; however, after treatment with high doses a secretory-like activity is observed, accompanied with ultrastructural changes.Supported by the Deutsche Forschungsgemeinschaft, Special Program on Biology and Clinics of Reproduction. (Grant Be 524/7-7).Visiting Scientist from the Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel.     

Amino acid sequence of rabbit ventricular myosin light chain-2: Identity with the slow skeletal muscle isoform

Many studies have established a correlation of differences in the activities of various muscle types with differences in the expression of myosin isoforms. In this paper we report the sequence determination of myosin light chain-2 from rabbit slow skeletal (LC2s) and ventricular (LC2v) nmscles. We sequenced tryptic peptides from LC2v which account for all except a few terminal amino acid residues. The major part (87 residues) of the rabbit LC2s sequence, obtained from tryptic and cyanogen bromide (CNBr) peptides, was found to be identical to rabbit LC2v. Our results provide the first sequence information on LC2s from any species, and lend strong support to the hypothesis that LC2s and LC2v are identical. Comparisons of rabbit LC2v and LC2s with rabbit LC2f (from fast skeletal muscle), and also with chicken LC2f and LC2v, show clearly that LC2s and LC2v from mammalian and avian species are more closely related to each other than they are to LC2f isoforms from the same species.     

The Circadian Rhythm of Plasma Thyrotropin in Depression and Recovery

The 24-hr patterns of plasma thyrotropin have been observed in 12 endogenous depressed patients in both depressed and recovered states and in 13 normal subjects. A clear circadian rhythm was detected in controls with high values at night. In depression, the circadian rhythm was altered with amplitude reduction and blunted nocturnal secretion, abnormalities particularly relevant in bipolar patients. This flattened profile could be linked to the blunted response of TSH to TRH administration reported in depressed patients. Normal nyctohemeral patterns have been restored after recovery. These chronobiological abnormalities as well as their normalization under antidepressant drugs seem to be similar to those reported for various parameters (e.g. temperature, Cortisol, etc) in depression which could support the chronobiological hypothesis for affective disorders.