No description available.

Prolactin receptor (PRLR) is a membrane anchored protein mediating the biological actions of prolactin. The turkey PRLR (tPRLR) cDNA was isolated and characterized. The open reading frame (ORF) of tPRLR predicted 831 amino acid residues composed of a signal peptide, an extracellular domain (ECD), a single transmembrane domain and an intracellular domain. The deduced amino acid sequence of the turkey prolactin receptor is 53.8%, 51.7%, 49.8%, 49.8%, 80.3% and 89.91% identical to that of the rabbit bovine, human, long form of the rat, pigeon and chicken PRLRs, respectively. The extracellular domain contains two homologous repeat units with 63% amino acid sequence identity to each other. The membrane-distal and membrane-proximal repeats were 53--60% and 62--70% identical to the ECDs of the mammalian PRLRs, respectively. A tPRLR transcript with a molecular size of 3 kilo nucleotides was identified and was detectable in 26 tissues examined. The pituitary gland, crop sac, duodenum and gizzard were found to express the highest levels of tPRLR mRNA among the 26 tissues. In most tissues examined there was no obvious relationship between blood levels of PRL, reproductive states and estimated concentrations of the receptor mRNA. However, in the hypothalamus, increasing blood levels of PRL were associated with decreasing levels of receptor transcripts (p < 0.05), whereas, the opposite relationship was observed in the pituitary gland (p < 0.05). The extracellular domain of tPRLR (tPRLR-ECD) was expressed as a GST fusion protein (tPRLR-ECD-GST) in E. coli. The expression of tPRLR-ECD-GST in BL21 strain yielded a protein with a molecular mass of 76 kDa. About 99% of the fusion protein was present in inclusion bodies and about 50% of the total protein in inclusion bodies was the fusion protein. The insoluble fusion protein was denatured, refolded and purified using GST affinity chromatography. The yield of the purified fusion protein was 20 mg per liter with an estimated p

No description available.

Changes in the pituitary content of prolactin (PRL) and in the ratio of immunoreactive forms of PRL were measured during a reproductive cycle in turkey hens. Low levels of PRL were observed in pituitary glands from sexually immature, out-of-lay and moulting hens. Higher levels were present during the egg laying period and the highest levels were detected in hens expressing incubating behaviour. Two immunoreactive bands of apparent molecular weights of 24 and 27 kDa were visualised on western blots, corresponding to the non-glycosylated (NG-) and glycosylated (G-) forms of PRL, respectively. The percentage of G-PRL was about 60 in sexually immature and egg laying hens. In pituitaries from incubating, out-of-lay and moulting hens the percentage of G-PRL was about 70, 38 and 33, respectively. Thus, higher percentages of G-isoforms (27 kDa) were associated with high levels of total PRL and Iower percentages were associated with low levels of PRL content in the pituitary gland. Digestion of the isoforms with N-glycosidase F resulted in a single band with an apparent molecular weight of 24 kDa. Partial deglycosylation was achieved using neuraminidase, whereas digestion with O-glycosidase had no apparent effect on the isoforms. Thus, G-PRL has N-linked carbohydrates containing sialic acid. In order to study the in vitro release of the PRL isoforms, pituitary glands from turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Total PRL content and the ratio of immunoreactive PRL isoforms in the perifusate were monitored. All the perifused pituitaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands corresponding to the ones detected in pituitary extracts were detected by western blotting. The G-PRL (27 kDa) was predominant in samples from egg laying and incubating hens and the NG-PRL (24 kDa) was predominant in samples from out-of-lay and moulting hens. No change in the ratio of isoforms released was / A competitive reverse transcription polymerase chain reaction assay was developed in order to semi-quantify the PRL mRNA in individual pituitary glands from turkey embryos and poults. Pituitary content and plasma levels of PRL were also monitored, and the PRL isoforms present in the pituitary gland were detected. The levels of PRL mRNA remained low until five days before hatching, increased until the day of hatch, plateaued during the first three days of age and significantly increased at two weeks of age. Similar changes were observed in pituitary content and plasma concentrations of PRL which were highly correlated. Two immunoreactive bands corresponding to the NG- and G-PRL detected in adult pituitary gland were visualised on western blots. The percentage of G-PRL in pituitary glands were 31.5, 48.6, 48.0 and 56.2 at 22 and 27 days of incubation and at I and 7 days of age, respectively. Thus, higher percentages of G-PRL (27 kDa) were associated with higher levels of total PRL in the pituitary gland.

No description available.

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

The Mac-T cell system was used to investigate the role of the prolactin (PRL) receptor in cell proliferation and the regulation of milk protein synthesis. This study was designed to investigate whether overexpressing the PRLR in the Mac-T cell line resulted in a change in its growth rate and an enhancement of its ability to produce milk proteins. To accomplish these goals, Mac-T cells were stably transfected with the rabbit prolactin receptor gene. Fifteen clones and a pool of transfectants were obtained. Of these, one clone and the pool were positive for the PRL receptor expression. The clone (S15) and pool (SP) cells were sorted into high (H), medium (M), and low (L) expressors, of the PRLR. The high expressors were used for all subsequent experiments. The presence of high levels of the PRLR on the surface of S15 and SP cells was further confirmed by receptor binding assay and Western Blot. Following the establishment of these cell lines, the cells were used to investigate the effect of increased levels of PRLR on cell proliferation and milk protein synthesis. / It was found that the growth rate of parental cells was depressed in the presence of 5 mug/ml of PRL. In contrast, the growth rate of the transfectants was enhanced by the addition of 5 mug/ml PRL to the culture medium. In addition, both "SP" and "S15" cells produced higher levels of STAT5 upon long-term (48 h) PRL stimulation. No effect on the synthesis of alpha S1- and beta-caseins was noted. It is likely that no differences in protein synthesis were observed because the cells have lost the ability to differentiate, even when cultured on collagen gels in the presence of lactogenic hormones.

A turkey prolactin (PRL) cDNA, encoding a 199 amino acid turkey PRL (tPRL), was cloned from a pituitary library. The mature PRL shared about 70% homology with mammalian PRLs and about 30% with fish PRLs. Areas of highest homology to other PRLs were located in the carboxyl terminus of the tPRL. Prolactin mRNA analyses, during the reproductive life of the turkey hen, confirmed that the high pituitary and plasma levels of PRL measured during the incubation phase are due to enhanced transcription of the PRL gene. Furthermore, tPRL mRNA levels were highly correlated with pituitary levels of tPRL. Recombinant tPRL (rctPRL), biologically and immunologically similar to pituitary tPRL, was purified from Escherichia coli cultures hosting an expression vector carrying the tPRL cDNA. Polyclonal antibodies raised against purified rctPRL behaved similar as antibodies raised against pituitary derived tPRL, in immunoblotting and immunocytochemistry experiments. Three tPRL isoforms (with estimated molecular weights of 27 kDa, 25 kDa and 24 kDa) were identified in turkey pituitary extracts. The relative proportion of the 27 kDa isoform increased while that of the 25 kDa decreased with increasing levels of total pituitary tPRL, during the reproductive life of the turkey hen. The partition of the immunoreactivity of tPRL into the three isoforms perhaps provides an additional control of the multitude functions of PRL.

No description available.