Ovine Peptide Research Articles

Hypothalamic Distribution, Adenohypophyseal Receptor Expression, and Ligand Functionality of RFamide-Related Peptide 3 in the Mare During the Breeding and Nonbreeding Seasons1

RFamide-related peptide 3 (RFRP3), the mammalian homologue of avian gonadotropin-inhibitory hormone, has been shown to negatively regulate the secretion of LH and may contribute to reproductive seasonality in some species. Herein, we examined the presence and potential role of the RFRP3-signaling system in regulating LH secretion in the mare during the breeding and nonbreeding seasons. Hypothalamic NPVF mRNA (the precursor mRNA for RFRP3) was detected at the level of the dorsomedial nucleus and paraventricular nucleus, but expression did not change with season. A greater number of RFRP3-expressing cells was observed throughout the rostral-caudal extension of the dorsomedial nucleus. Furthermore, adenohypophyseal expression of the RFRP3 receptor (NPFFR1) during the winter anovulatory season did not differ from that during either the follicular or luteal phases of the estrous cycle. When tested in primary adenohypophyseal cell culture or in vivo during both the breeding and nonbreeding seasons, neither equine nor ovine peptide sequences for RFRP3 suppressed basal or GnRH-mediated release of LH. However, infusion of RF9, an RFRP3 receptor-signaling antagonist, into seasonally anovulatory mares induced a robust increase in secretion of LH both before and following continuous treatment with GnRH. The results indicate that the cellular machinery associated with RFRP3 function is present in the equine hypothalamus and adenohypophysis. However, evidence for functionality of the RFRP3-signaling network was only obvious when an antagonist RF9 was employed. Because GnRH-induced release of LH was not affected by RF9, its actions may occur upstream from the gonadotrope to stimulate or disinhibit secretion of GnRH.

Kinome profiling of BLV-induced ovine leukemia: an approach for identifying altered signaling pathways associated with oncogenesis

Transcriptome and miRnome information will likely be of significant value to our elucidation of the molecular mechanisms that govern cell transformation. However, an equally, if not more important goal, is to define those proteins that participate in signaling pathways that ultimately control cell fate. Bovine Leukemia Virus (BLV), a delta-retrovirus closely related to HTLV-1, is associated with B-cell leukemia in sheep. We have employed kinome arrays which contain ovine peptide substrates selected to target known phosphorylation sites in proteins regulating key cell signaling pathways. Our data provide a quantitative measure of the phosphorylation activity of 300 kinases. We found significant changes in phosphorylation patterns of primary ovine leukemia/lymphoma versus normal B-cells. Pathway analysis tools revealed changes in proteins playing a major role in signaling cascades that determine cell-cycle entry, proliferation, survival and differentiation. Interestingly, analysis of cultured transformed B-cell lines suggested cell signaling events that characterize primary cancer cells were not conserved in vitro. Using NOD-Scid-Gamma immunodeficient mice and SC injection of ovine transformed B cell lines generated in vitro, we asked if transformed B-cells grown in mice would reflect the initial in vivo kinome profile identified in leukemic sheep. Finally, a priority identified for defining the rigour of our dataset was kinome analysis of an increased number of normal B-cells to provide an estimate of reference kinome diversity in an outbred population. Altogether, these investigations will provide a critical analysis of the utility of kinome arrays as a technology to analyze oncogenesis, identify therapeutic targets, and select potential cancer biomarkers.

Factors affecting the antimicrobial activity of ovine-derived cathelicidins against E. coli 0157:H7

Antimicrobial peptides extracted from ovine neutrophils have potential to be high-value by-products of the lamb industry as, for example, a biopreservative for chilled lamb products. This work was carried out to determine the conditions in which ovine peptides are most effective and to assist in product development. The activities of three synthetic ovine-derived antimicrobial peptides tested were not significantly affected by pH or temperature. However, they exhibited decreased activity at high ionic strengths and in the presence of divalent cations. The three peptides worked better in combination than individually.

The characterization of ovine genes for atrial, brain, and C-type natriuretic peptides

The natriuretic peptides (NPs) play an important role in the homeostasis of blood pressure and sodium balance in all mammals studied to date. Their combined actions on the vasculature, kidneys, and adrenals reduce blood pressure and intravascular volume. In order to provide sequence information about the ovine NP genes for our physiological studies in sheep, we have determined the genomic DNA sequence of each of the NPs; atrial NP, brain NP (BNP), and C-type NP using an ovine genomic library. Strong homology with other species was found for ovine peptide and genomic sequences of atrial NP and for C-type NP. Further, despite previous reports of poor conservation of BNP across species, the peptide sequence for ovine BNP was found to be identical to both the 26 amino acid-residue porcine BNP, and the 35 amino acid peptide known as bovine aldosterone secretion-inhibitory factor. This data also revealed strong homology of BNP mature forms in dog, cow, pig, and sheep, thus permitting the use of porcine antisera to study BNP-level changes in sheep models of cardiac failure. This conservation of the BNP gene sequence suggests that BNP, like the other NPs, plays an important role in mammalian physiology.

Corticotropin-releasing factor administered centrally, but not peripherally, stimulates hippocampal acetylcholine release.

In addition to corticotropin-releasing factor's well-known role in mediating hormonal and behavioral responses to stress, this peptide also reportedly affects arousal and cognition, processes that classically have been associated with forebrain cholinergic systems. Corticotropin-releasing factor stimulation of cholinergic neurons might thus provide a mechanism for this peptide's cognitive effects. To examine this possibility, the present experiments characterize the effect of corticotropin-releasing factor on cholinergic neurotransmission, using in vivo microdialysis to measure hippocampal acetylcholine release. Corticotropin-releasing factor (0.5-5.0 microg/rat intracerebroventricularly) was found to increase dialysate concentrations of acetylcholine in a dose-dependent manner in comparison with a control injection, the ovine peptide having a greater effect than the same dose of the human/rat peptide. This effect was found to be centrally mediated, independent of the peripheral effects of an exogenous corticotropin-releasing factor injection; subcutaneous injections of the peptide increased plasma concentrations of corticosterone, the adrenal hormone ultimately secreted in the rat's stress response, to the same level as did the central injections, without affecting hippocampal acetylcholine release. These results demonstrate that corticotropin-releasing factor, acting centrally, regulates hippocampal cholinergic activity, and suggest that corticotropin-releasing factor/acetylcholine interactions may underlie some of the previously identified roles of these neurotransmitters in arousal, cognition, and stress.

Characterization of a sheep brain corticotropin releasing factor binding protein

We report here the identification, purification and cDNA cloning of a corticotropin releasing factor (CRF) binding protein(s) (CRF-BP) from sheep brain. Native sheep and rat brain CRF-BP and recombinant rat CRF-BP were shown to be N-glycosylated. Two membrane associated forms of brain CRF-BPs of 33 and 35 kDa were purified from sheep brain homogenates after solubilization in the presence of detergent. N-Terminal sequence analysis revealed that the 35 kDa protein is proteolytically cleaved near the N-terminus giving rise to an 18 amino acid peptide and a 33 kDa CRF-BP. Both the purified 33 and 35 kDa ovine CRF-BPs could be specifically cross linked to ovine [ 125I]CRF and human [ 125I]CRF. In contrast, recombinant rat CRF-BP can only be cross-linked to human [ 125I]CRF. A 1.7 kb cDNA clone (Basil 7) encoding an open reading frame for a 324 amino acid CRF-BP precursor was cloned from a sheep brain lambda gt10 cDNA library and was shown to have 85% and 87% amino acid homology to the rat and human proteins, respectively. Competitive binding analysis of the recombinant sheep CRF-BP (Basil 7) expressed in CHO cells revealed that it binds human and ovine CRF with high affinity. However, the recombinant sheep CRF-BP (Basil 7) had approximately 50-fold higher affinity for human CRF than for the ovine peptide. These data present the first biochemical proof that CRF-BP is in the brain and provides evidence for the existence of different forms of CRF-BP which have evolved across species to regulate CRF.

Corticotropin Releasing Factor (CRF) Binding Protein: A Novel Regulator of CRF and Related Peptides

A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding protein's ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF-BP is an important modulator of CRF both in the CNS and in the periphery.

Growth hormone-releasing factor from ovine and caprine hypothalamus: Isolation, sequence analysis and total synthesis

Peptides with high intrinsic growth hormone releasing activity (growth hormone-releasing factor, GRF) were isolated from 2100 ovine and 2600 caprine (goat) hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration and reverse phase HPLC. Structural characterization of the 44 amino acid ovine peptide by gas-liquid phase sequencing and peptide mapping established its primary structure as Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-NH 2. Caprine GRF was found to possess the same sequence except for the replacement of the isoleucine residue in position 13 with valine and thus is identical to bovine GRF.

Adrenal medullary chromaffin granule peptides: Two major ovine peptides and their precursor

An octapeptide and decapeptide which are not derived from proenkephalin were isolated from ovine adrenal chromaffin granules. Their sequences are AsnLeuAspProLysLeuAsp Leu and ValAlaGluLeuAspGlnLeuLeuHisTyr. These two peptides were found to be derived from a single precursor peptide which has also been isolated and sequenced. The proteolytic cleavage occurs at a Lys-Arg site typical of prohormone to hormone cleavages.

The Neurohypophysial Hormone‐Binding Proteins: Complete Amino‐Acid Sequence of Ovine and Bovine MSEL‐Neurophysins

Ovine and bovine MSEL‐neurophysins, purified by hormone protein complex precipitation, molecular sieving and ion‐exchange chromatography, were subjected either to performic acid oxidation or to reduction and carboxamidomethylation. Cleavage by trypsin gave in each case eight fragments which were separated by peptide mapping. They were sequenced either directly by Edman degradation or after sub‐cleavage by subtilisin, chymotrypsin, thermolysin, pepsin or partial acid hydrolysis and characterization of the sub‐peptides.Alignment of the tryptic fragments was determined by automated Edman degradation and by identification of overlapping peptides obtained after hydrolysis of the polypeptide chain by chymotrypsin, pepsin and subtilisin. Bovine tryptic peptides were arranged, in part, according to their homology with ovine peptides.Both ovine and bovine MSEL‐neurophysins comprise 95 residues; in both cases, a three‐residue N‐terminal truncated form can be detected. Ovine and bovine MSEL‐neurophysins are nearly identical. Two positions are different: 48 (isoleucine in ovine and asparagine in bovine) and 89 which shows a microheterogeneity in the ox (isoleucine in ovine. isoleucine or valine in bovine). Porcine MSEL‐neurophysin presents four differences when compared with ovine homologue (positions 48, 89, 90 and 92) and an apparent three‐residue C‐terminal deletion.MSEL‐neurophysins form a protein family which appears to be evolutionary stable in species of Artiodactyla. In this order of mammals, the rare variations seem to occur in the C‐terminal sequence. A second family of related neurophysins found in mammals. VLDV‐neurophysins, shows substitutions in the N‐terminal sequence when compared to MSEL‐neurophysins. There is a large invariant central part in the polypeptide chain which might include the neurophysial hormone‐binding domain of all neurophysins.