Epitope mapping and analysis of a growth-enhancing monoclonal antibody by limited tryptic digestion of porcine GH.

In this study, the epitope of a murine PS-7.6 monoclonal antibody (mAb) which was raised against the recombinant porcine GH (pGH) and subsequently shown to enhance the growth-promoting activity of pGH in a hypophysectomized rat model, was mapped by the limited tryptic digestion of pGH. A pGH fragment corresponding to amino acid residues 70-95 was separated by reverse-phase HPLC and also immunoprecipitated by PS-7.6 mAb. This fragment was found in an RIA to compete with radiolabelled pGH for the binding of PS-7.6 mAb in a dose-dependent fashion. Several peptides covering this potential epitope region of pGH(70-95) were synthesized and assayed by competitive RIA. The results suggested that pGH(75-90) was the optimal sequence recognized by PS-7.6 mAb. Sequential alanine substitution of each residue of pGH(75-90) revealed that the side chains of Leu76, Ile83 and Leu87 were critical for binding to PS-7.6 mAb. Other residues could be replaced by alanine without substantially altering the binding affinity. The region of amino acids 75-95 comprises the C-terminal end of the second helix of pGH and the repeating pattern of i and i + 3 (i + 7) of the critical amino acids appears consistent with PS-7.6 mAb binding to the hydrophobic side of the helix. The sequence and the helical structure of the epitope of PS-7.6 mAb provide the basis for designing the effective peptide vaccines to enhance the growth performance of animals.

Serum immunoglobulin type G concentrations in calves produced by IVF and delivered by elective cesarean section.

Colostrum ingestion by neonatal calves is widely recognized to provide passive transfer of immunity. In this study immunoglobulin absorption from colostrum was evaluated in 54 IVF-produced calves. The IVF calves were delivered by Cesarean section on Days 275 to 277 of gestation, 24 h after the dams had been administered 30 mg dexamethasone. The calves suckled bottles or were force-fed 6 L of colostrum in the first 12 h of life. Colostrum was obtained from the first post-calving milking of recipient dams or from frozen storage reserves if dam secretion was not adequate. Immunoglobulin type G (IgG) content of both sources of colostrum was determined. Serum samples from the calves were collected at 0, 12 and 24 h of age and analyzed for IgG. Twenty dairy calves born vaginally served as the controls and were subjected to the same colostrum management protocol except that the colostrum was obtained only from frozen post-calving milk of dairy cows from the same farm. The control calves were also subjected to the same sampling protocol. The IVF group of calves ingested more IgG (P < 0.0001) and absorbed more IgG by 24 h of age (P < 0.0001) than their control group counterparts. Absorption of IgG was analyzed by comparing the g/kg body weight of IgG with serum IgG values at corresponding times after birth. Colostrum absorption efficiency was the same for both IVF and control groups of calves at 12 and 24 h of age. There was a maximum IgG dose above which additional increases in serum IgG were not realized. The slightly premature, Cesarean delivered IVF calves absorbed IgG from colostrum similarly to control calves delivered vaginally.

Effects of oral vitamin E supplementation during late gestation in beef cattle that calved in late winter and late summer.

OBJECTIVE: To determine effects of breed and oral vitamin E supplementation during late gestation on serum vitamin E and IgG concentrations in beef cows that calved in late winter and late summer and in neonatal calves. ANIMALS: 73 Angus and 43 Hereford primiparous and multiparous cows and their calves. PROCEDURE: Cows in groups that were homogeneous regarding breed and age distribution were randomly allotted to groups that were orally supplemented (n = 59) or not supplemented (57) with vitamin E beginning 30 days prior to onset of 65-day calving seasons. Supplemental vitamin E was provided in a vitamin-mineral mix offered free-choice until parturition. RESULTS: Cows that calved in late winter and were supplemented orally with vitamin E had higher serum vitamin E concentrations at calving and after calving than did unsupplemented cows; differences between groups before calving were not significant. Calves from supplemented multiparous cows had higher vitamin E concentrations than did calves from unsupplemented cows. Winter-born calves from supplemented Hereford cows had heavier 205-day adjusted weaning weights than did winter-born calves from unsupplemented Hereford cows. Supplementation did not affect vitamin E or IgG concentrations in the herd that calved in late summer and did not affect calf growth. CONCLUSIONS AND CLINICAL RELEVANCE: Oral vitamin E supplementation during late gestation may be economically beneficial in certain cow-calf operations in which late-gestation cows are consuming stored forages.

Effects of supplemental parenteral administration of vitamin E and selenium to Jerseys and Holsteins during the nonlactating period.

OBJECTIVE: To determine effects of breed and supplemental administration of vitamin E and selenium (Se) during late gestation on circulating concentrations of these micronutrients in periparturient Jerseys and Holsteins. DESIGN: Randomized controlled clinical study. ANIMALS: 16 Jersey and 36 Holstein cows. PROCEDURE: Cows were allotted to blocks on the basis of breed and expected parturition date. Cows within blocks were randomly assigned to be given vitamin E or Se parenterally 3 to 4 weeks prior to anticipated parturition in a 2 x 2 factorial design. RESULTS: Results of ANOVA indicated Jerseys had higher blood concentrations of Se and lower serum concentrations of vitamin E than Holsteins at the end of lactation. Jerseys had higher blood concentrations of Se than Holsteins 3 to 4 weeks prior to parturition and at parturition. Selenium administration increased blood concentrations of Se at parturition. Administration of nutrients did not affect serum concentrations of vitamin E at parturition or 2 to 3 weeks after parturition or blood concentrations of Se 2 to 3 weeks after parturition. CONCLUSIONS AND CLINICAL RELEVANCE: Jerseys and Holsteins consuming rations of comparable Se content differ in blood concentrations of Se during the nonlactating period, suggesting breed-related differences in Se metabolism during late lactation and the nonlactating period. Parenteral administration of Se 3 to 4 weeks prior to anticipated parturition increased blood concentrations of Se at parturition; however, Se concentrations of both groups at parturition were considered within the reference range for clinically normal cattle.

Identification and characterization of novel somatostatin antagonists.

The study of the five somatostatin receptor subtypes (SSTx, where x is the subtype number) has been hampered by the lack of high affinity antagonists. Potent and selective antagonists would increase our understanding of SST structure, function, and regulation. In this study, the identification of novel disulfide-linked cyclic octapeptide antagonists of somatostatin is described. The antagonists contain a core structure of a DL-cysteine pair at positions 2 and 7 of the peptides. Substitution of a D-cysteine at position 2 with an L-cysteine converts the full antagonist into a full agonist. All somatostatin receptor subtypes are coupled to inhibition of adenylate cyclase. The functional properties of these peptides have been determined in radioligand binding assays, in functional coupling of the SST2 subtype to yeast pheromone response pathway, and in cAMP accumulations. One peptide antagonist [Ac-4-NO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-D-Tyr-NH2] displays a binding affinity to SST2 comparable with that observed for the native hormone (Ki = 0.2 nM) and reverses somatostatin-mediated inhibition of cAMP accumulation in rat somatomammotroph GH4C1 cells, cells transfected with the SST2 and SST5 subtypes, as well as somatostatin-stimulated growth of yeast cells expressing the SST2 subtype. This class of somatostatin antagonists, which are the first to be described, should be useful for determination of somatostatin's diverse functions in vivo and in vitro.