Preparation and characterization of recombinant chicken growth hormone (chGH) and its putative antagonist chGH G119R mutein.

Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized after being optimized for expression in E. coli. The respective cDNAs were inserted into expression vector, expressed and found almost entirely in the insoluble inclusion bodies (IBs). The IBs were isolated, the proteins solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded, and purified to homogeneity by anion-exchange chromatography on Q-Sepharose. The overall yields were 400 to 500 mg from 5 L of fermentation. Both proteins were > 98% pure, as evidenced by SDS-PAGE, and contained at least 95% monomers, as documented by gel-filtration chromatography under non-denaturing conditions. Circular dichroism analysis revealed that both proteins have identical secondary structure characteristic of cytokines, namely > 50% of alpha helix content. Chicken GH was capable of forming a 1:2 complex with recombinant oGH receptor extracellular domain, but its affinity, as determined by RRA, was 11-fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed using FDC-P1 3B9 cells stably transfected with rabbit GHR, was 30-40-fold lower, whereas chGH G119R mutant did not bind to oGHR-ECD and was devoid of any biological activity in FDC-P1 3B9 cells. However, in binding experiments that were carried out using chicken liver membranes, both oGH and chGH showed similar IC(50) values in competition with (125)I-oGH, while the IC(50) of G119R mutein was 10-fold higher. These results emphasize the importance of species specificity and indicate the possibility of antagonistic activity of chGH G119R.

Immunological effect of a synthetic growth hormone peptide on the growth performance in swine.

A mouse monoclonal antibody (mAb), designated PS-7.6, was previously shown to enhance the activity of porcine growth hormone (pGH) in promoting the growth of hypophysectomized (hypox) rats. Epitope mapping studies indicated that the region recognized by PS-7.6 resided within an amino acid sequence 54-95 of pGH. A peptide corresponding to this sequence was synthesized and found to induce swine antibodies capable of augmenting pGH activity in hypox rats. On the basis of these previous observations, an attempt was made in this study to determine whether or not the peptide pGH(54-95) could be used as a vaccine to elicit antibodies functionally similar to PS-7.6 mAb, thus potentiating the efficacy of endogenous GH in swine. Young pigs (15-20 kg) were immunized with pGH(54-95) that had been conjugated with ovalbumin (OVA) and boosted twice at 4-week intervals. Control animals were similarly immunized with OVA. The weight gains and feed consumption of these animals were closely monitored throughout the trials. A number of carcass parameters were also examined when these animals reached 110-120 kg, at which time they were killed. Results indicated that immunization with peptide significantly accelerated the daily weight gain during the growing phase of growth. However, this effect disappeared during the finishing phase of growth. The failure to prolong the initial growth effect by the peptide immunization apparently correlated with the kinetics of antibody production, because antibodies immunoreactive to the peptide and pGH were detected in these animals after immunization but gradually diminished. This idea was supported by the fact that antibodies obtained from pigs 5 and 9 weeks after the initial immunization potentiated the activity of pGH in hypox rats, whereas antibodies harvested at week 16 did not. Furthermore, carcass evaluation was performed at time of killing and showed that the leaf fat and loin eye muscle were also significantly improved by peptide immunization. Taken together, the present findings suggest that pGH(54-95) peptide can be employed as a potential growth-promoting vaccine to improve the performance of swine.

Conjugation of synthetic peptides to carrier iscoms: factors affecting the immunogenicity of the conjugate.

This study was designed to explore optimal conditions for the conjugation of a synthetic peptide to preformed influenza virus iscoms using MHS (maleimidohexanoyl-N-hydroxy-succinimide ester) as coupling agent. The peptide used in this study comprised amino acids 122-138 of porcine growth hormone (pGH). Different ratios of peptide to carrier iscoms were tested and the resulting conjugates were analysed for composition, antigenicity and immunogenicity. The problem of low solubility and poor immunogenicity of high-density peptide conjugates is discussed and a general protocol for conjugation of peptides to carrier iscoms is proposed.

The effects of synthetic growth hormone on spinal cord injury.

The aim of this study was to determine the effect of synthetic growth hormone on healing process of reversible injured spinal cord. The rat was selected for the experiment in preference to larger animals for economy and availability. Under general anesthesia one level laminectomy was carried out at T-1 with the dura mater intact. The injury was created by Rivlin and Tator's clip method. Recovery of motor function was assessed for up to 4 weeks using inclined plane test of hind limb motor function. Although the effect of the drug is evident after 3 weeks, the study must be held for longer periods for a sufficient regeneration time to maintain a significant statistical evaluation.

Development, production and pharmacodynamics of human growth hormone.

With the advent of recombinant DNA technology, it is possible to produce biosynthetic human growth hormone (B-hGH). Novo Nordisk A/S has developed a method for manufacturing B-hGH which is identical to the 22K fraction of pituitary human growth hormone (P-hGH), using a nonpathogenic strain of Escherichia coli as host. B-hGH has been investigated extensively in physical, chemical and biological studies and found to be identical to P-hGH. Pharmacological studies have revealed that B-hGH possesses the same pharmacokinetic and short-term metabolic profiles as P-hGH. Long term clinical studies have shown that B-hGH induces a significant increase in height velocity in children with growth hormone deficiency (GHD) and is characterized by a low antigenicity.

Sorption and mineral-promoted transformation of synthetic hormone growth promoters in soil systems.

This work examines the fate of synthetic growth promoters (trenbolone acetate, melengestrol acetate, and zeranol) in sterilized soil systems, focusing on their sorption to organic matter and propensity for mineral-promoted reactions. In organic-rich soil matrices (e.g., Pahokee Peat), the extent and reversibility of sorption did not generally correlate with compound hydrophobicity (e.g., K(ow) values), suggesting that specific binding interactions (e.g., potentially hydrogen bonding through C17 hydroxyl groups for the trenbolone and melengestrol families) can also contribute to uptake. In soils with lower organic carbon contents (1-5.9% OC), evidence supports sorption occurring in parallel with surface reaction on inorganic mineral phases. Subsequent experiments with pure mineral phases representative of those naturally abundant in soil (e.g., iron, silica, and manganese oxides) suggest that growth promoters are prone to mineral-promoted oxidation, hydrolysis, and/or nucleophilic (e.g., H2O or OH(-)) addition reactions. Although reaction products remain unidentified, this study shows that synthetic growth promoters can undergo abiotic transformation in soil systems, a previously unidentified fate pathway with implications for their persistence and ecosystem effects in the subsurface.

Highly potent growth hormone secretagogues.

During an effort to search for more potent growth hormone secretagogues, we discovered a class of compounds of which the best compound 8 was 7-fold more active in vitro than the best compound in the series we revealed before [Tata, J. R.; Lu, Z.; Jacks, T. M.; Schleim, K. D.; Cheng, K.; Wei, L.; Chan, W.-S.; Butler, B.; Tsou, N.; Leung, K.; Chiu, S.-H. L.; Hickey, G. J.; Smith, R. G.; Patchett, A. A. Bioorg. Med. Chem. Lett.1997, 7, 2319.]. Animal studies show that compound 8 can stimulate growth hormone release at the oral dose as low as 0.06 mpk. Chemistry and biological studies are discussed.

Solution conformation of a synthetic fragment of human pituitary growth hormone. Two-dimensional NMR of an alpha-helical dimer.

Circular dichroism and two-dimensional NMR spectra indicate that a peptide fragment consisting of the first 28 residues from the N-terminus of human growth hormone (hGH 1-28) has considerable alpha-helical structure. The peptide, (1) H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-Hi s-Arg- Leu-His-Gln-Leu-Ala-Phe-Asp-Thr-Tyr-OH (28), was synthesized on an automated peptide synthesizer using the Merrifield solid-phase method. The peptide can be modeled as an amphiphilic helix, and the unusual stability of the alpha-helix in aqueous solution is suggested to be attributable to formation of a dimer of alpha-helices. Most of the 1H NMR signals were assigned through pure absorption phase COSY/NOESY and single- and double-relay COSY 2D NMR spectra by using the sequential assignment methodology. The NOEs were large and negative, suggesting that the peptide was not a random coil and that it existed in solution primarily as a large, fairly rigid macromolecule, consistent with the dimer structure. A network of N alpha Hi-N alpha Hi+1 NOESY crosspeaks is observed from residues 13 to 18 as are several other crosspeaks which indicate that the peptide has considerable alpha-helical structure between residues 8 and 24. In addition, gel filtration of the peptide is consistent with a dimer structure, presumably involving packing of the two hydrophobic faces of the amphiphilic alpha-helices.

Diabetogenic activity of native and biosynthetic human growth hormone in obese (ob/ob) mouse.

It has been repeatedly suggested that diabetogenic activity is not an intrinsic property of native pituitary growth hormone (GH) and that the diabetogenic effects produced by GH preparations are due to low-molecular-weight contaminants or degradation products of the hormone. This possibility was evaluated in this study by assessing the ability of purified native human GH (hGH) and biosynthetic methionyl-hGH to exacerbate fasting hyperglycemia and glucose intolerance in the obese (ob/ob) mouse. Native hGH that had been purified by DEAE-cellulose chromatography (A-type; 1.8 IU/mg) produced fasting hyperglycemia and glucose intolerance in the ob/ob mouse when injected subcutaneously at doses of 50 micrograms/day or greater for 3 days. It had no effect when a single subcutaneous dose of 200 micrograms was administered 24 h previously. To eliminate possible contamination with smaller peptides, the hGH was gel-filtered on a column of Sephacryl S-200 in 6 M guanidine-HCl. When injected subcutaneously into ob/ob mice at a dose of 50 micrograms/day or greater for 3 days, the guanidine-treated hGH produced glucose intolerance. Also biosynthetic methionyl-hGH produced marked fasting hyperglycemia and glucose intolerance when injected subcutaneously at doses of 50 or 100 micrograms/day for 3 days. These results support the conclusion that hGH itself is indeed diabetogenic but that chronic exposure of the organism to the hormone is required for its effects on glucose metabolism to become clearly manifest.

Activity of artificial mutant variants of human growth hormone deficient in a disulfide bond between Cys53 and Cys165.

In order to understand the role of Cys53 and Cys165 of human growth hormone (hGH) in receptor-binding and biological activity, artificial mutant variants of hGH were prepared in Escherichia coli by in vitro mutagenesis. Variants of hGH were constructed by replacement of Cys165 with Ala ([Ala165]hGH) or Ser ([Ser165]hGH), by replacement of Cys53 with Ala ([Ala53]hGH), by replacement of Cys53 and Cys165 with Ala ([Ala53, Ala165]hGH), or by replacement of Cys53 with Ala and Cys165 with Ser ([Ala53,Ser165]hGH). All of the variants constructed as well as reduced hGH exhibited less biological activity than that of intact hGH, and the decreases in biological activity were almost equal, as measured by a sensitive biological assay for growth hormone: adipose conversion assay using 3T3-F442A cells. These variants also showed less receptor-binding activity than that of intact hGH. These results suggest that it is possible neither the residue Cys53 nor Cys165 is directly involved in the receptor binding, and that the disulfide bridge between Cys53 and Cys165 in hGH may not always be crucial for the biological activity, though necessary to express full hGH activity.

Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone.

Two derivatives of biosynthetic human growth hormone, a sulfoxide and a mixture of two monodesamido isomers, have been isolated and characterized. The sulfoxide derivative arises from an oxidation of Met-14. The major site of deamidation is at Asn-149 with a minor site at Asn-152. In addition, a fraction has been isolated from a sample of human growth hormone that was maintained at 40 degrees C for 2 weeks. This fraction, the isolated impurities fraction, contains the sulfoxide and the desamido forms, thereby demonstrating that these derivatives are the primary degradation products of biosynthetic human growth hormone. The sulfoxide, the desamido, and the isolated impurities fraction exhibit full biological activity.

Human somatotropin 53. Synthesis and biological activity of the amino terminal fifty-four residue fragment.

The amino terminal 54 residue peptide fragment of human somatotropin [Cys(Gam)53]-HGH-(1-54), has been synthesized by the solid-phase method. The symmetrical anhydride and active ester coupling methods were used exclusively. The synthetic product was purified by gel fitration isoelectric focusing, and partition chromatography. It was found to be homogeneous by six additional criteria. In complement fixation experiments the synthetic product was immunologically active with antisera to HGH and [cys(Cam)53]-HGH-(134). Antiserum raised against the synthetic product was immunologically active in the homologous assay and with HGH,[Cys(Cam)53]-HGH-(1-134), and [Cys(Cam)53]-HGH-(15-125). The synthetic fragment exhibited 53% of the activity of [Cys(Cam)53]-HGH-(1-134) in the rat tibia assay.

Synthesis and properties of human growth hormone fragments.

The solid phase synthesis of fragments 75-120-NH2 and 73-128-GlyNH2 of the human growth hormone sequence is described. Purification was by solvent extraction, gel filtration and partition chromatography. No growth-promoting activity was detected in any of the peptides by the rat body-growth assay. An immunochemically reactive area was located in the sequence 73-128 when tested by complement fixation assays.

Diabetogenic action of human growth hormone. Synthesis and activity of C-terminal fragments.

Three peptides corresponding to the C-terminal region of human growth hormone have been synthesized by the solid-phase method: HGH-(177--191), HGH-(178--191) and HGH-(179--191). The diabetogenic activities of these synthetic peptides are reported. The data indicate that extension of HGH-(179--191) at its NH2-terminus is required for in vivo activity. The reduced and S-carbamidomethylated form of HGH-(177--191) was also active, indicating that the disulphide bond is possibly not a prerequisite for biological activity.