Jacob S. Tou

Isolation of Genomic Sequence Encoding a Biologically Active Bovine TGF- α Protein

AbstractGenomic clones encoding bovine TGF-α were identified by hybridization with probes derived from human TGF-α sequence. Nucleotide sequence of the clones predicts that mature bovine TGF-α is a 50 amino acid polypeptide which shares 96% and 92% homology with human and rat TGF-α, respectively. Bovine TGF-α with the predicted sequence was chemically synthesized and tested for activity. Synthetic bovine TGF-α competes in a radioreceptor assay with labelled mouse EGF with activity parallel to that of human TGF-α and mouse EGF. The mitogenic activity of bovine TGF-α is comparable to that of human EGF in causing proliferation of bovine mammary epithelial cells. An ∼ 5.0 kilobase RNA transcript is observed in polyadenylated RNA from MDBK cells by Northern blot analysis. The polymerase chain reaction detects the presence of a TGF-α transcript in many bovine tissues. These data indicate that bovine TGF-α may be a normal regulator of cell growth in the bovine animal.

Amphiphilic growth hormone releasing factor (GRF) analogs: Peptide design and biological activity in vivo

The first twenty-nine amino acids of human Growth Hormone Releasing Factor (hGRF) possess a distinct amphiphilic character. This is seen as twisted hydrophobic and hydrophilic bands in the helical net projection. Four amidated analogs were designed by optimizing amphiphilic and helical potentials of the native sequence. These designed analogs, with up to eight-amino acid changes, were tested in sheep via intravenous injection. The growth hormone-stimulating activities of the analogs were significantly higher when compared to bovine Growth Hormone Releasing Factor (bGRF44-NH2). This suggests that the amphiphilic conformation of GRF(1-29) is important to the receptor.

Chemical synthesis of bovine transforming growth factor-α: Synthesis, characterization and biological activity

Bovine transforming growth factor-alpha (bTGF-alpha) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-alpha was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-alpha competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-alpha. Compared to hEGF, bTGF-alpha elicits a greater response in a bovine mammary cell proliferation.

Stimulation of somatotropin secretion following peripheral administration of the tripeptide, syndyphalin 33 in sheep, pigs and rats

In the present study, a simple tripeptide alkylamine, syndyphalin 33 (SD33, Tyr-DMet (O)-Gly-methylphenethylamide) was shown to stimulate somatotropin (GH) secretion in sheep, hogs and rats following peripheral administration. Intravenous (i.v.) administration of SD33 at doses of 0.05, 0.1 and 0.2 mumol/kg stimulated a significant increase in circulating GH levels in sheep within 5 minutes post-injection. This response was not attenuated following repeated i.v. injections of SD33 (0.05 /mmol/kg) administered at 2 hour intervals. In addition, plasma GH levels were significantly stimulated following either subcutaneous (s.c.) or oral administration of SD33 in hogs and rats. Subcutaneous administration of SD33 at doses of 0.5, 1.0 and 2.0 mumol/kg stimulated a significant increase in plasma GH concentrations within 30 minutes of injection in both species. Oral administration of SD33 at 1.0, 10 or 100 mumol/kg in rats resulted in a significant elevation in plasma GH levels which peaked at 30 minutes post-gavage. In the pig, circulating GH levels were significantly increased within 30 minutes post-ingestion and remained elevated for at least 2 hours at the 2.0 mumol/kg dose level. The ability of naloxone to block SD33-stimulated GH secretion suggests that this peptide acts via mu opiate receptors.

Two Novel Bovine Somatotropin Species Generated from a Common Dehydroalanine Intermediate

Under stressed conditions such as prolonged exposure to high pH, the C-terminal disulfide bridge in bovine somatotropin (bST) is susceptible to a base catalyzed β-elimination reaction. This reaction converts the disulfide bond to a dehydroalanine residue with loss of a sulphur atom. Two altered species were isolated in pure form and determined to be generated from this dehydroalanine intermediate. One is a monomeric lanthionyl bST (L-bST) with a thioether linkage, and the other is an inter-molecular disulfide linked dimer containing a lysinoalanine. These two novel structures were unambiguously determined using various techniques including enzymatic digestion, amino acid sequencing and analysis, and mass spectrometry. The monomeric L-bST was demonstrated to be equipotent to normal bST in a hypox rat assay, thus showing that formation of lanthionine in place of this disulfide bond does not affect it bioactivity.

Structural characterization of the two refold dimers of recombinant bovine somatotropin (bST).

Two major dimers are generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin (bST). These dimers represent the major part of the inactive high molecular weight species that are formed in this process. The structures of the two dimers are unambiguously determined by peptide mapping using trypsin, thrombin cleavage, and selective DTT reduction experiments. Results indicate that the formation of both dimers involves the large disulfide loop cysteines. The latter-eluting dimer from RP-HPLC, previously reported as a large loop concatenated dimer, was revised to be an antiparallel disulfide-linked dimer. On the other hand, the first eluting dimer is a concatenane in which two monomers are held together by the interlocking of the two large disulfide loops.