Gwen G. Krivi

Cloning and in vivo expression of bovine growth hormone receptor mRNA.

A cDNA for the bovine growth hormone (bGH) receptor has been cloned out of a cDNA library prepared from liver of a pregnant Holstein heifer. The cDNA clone hybridizes to a single 4.5 kb mRNA species and shares a high degree of sequence homology with growth hormone receptors cloned from other species. Utilizing the bGH receptor cDNA as a probe, a relatively high level of bGH-receptor mRNA was detected in bovine liver. In comparison to liver values, lower concentrations of bGH-receptor mRNA were detected in bovine kidney, anterior pituitary, and mammary gland. Because specific binding sites for bGH have not been convincingly demonstrated in isolated cell membranes from whole bovine mammary tissue, mammary tissue from two pregnant heifers (separate experiments) was separated into fractions enriched for epithelium, stroma, and blood components. These fractions were then probed for growth hormone receptor mRNA using solution hybridization-nuclease protection assays performed on isolated RNA. The assay results indicated that a low level of bGH-receptor mRNA is relatively evenly distributed throughout the mammary tissues of the two cows studied. In contrast, experiments using a probe to bovine insulin-like growth factor-I (IGF-I) indicate that the IGF-I mRNA is localized in the stromal/blood component of the mammary gland. These data suggest a possible paracrine mechanism for bGH action in the mammary gland.

Variants of somatotropin in cattle: gene frequencies in major dairy breeds and associated milk production

The amino acid sequence of bovine somatotropin (bST) varies at position 127 where either valine or leucine is found. The frequencies of leucine127 and valine127 bST gene alleles in cows (n = 302) and sires (n = 70) from major dairy breeds (Holstein, Brown Swiss, Guernsey, Jersey, and Ayrshire) were determined using DNA extracted from whole blood or spermatozoa. A 428 base pair fragment of the bST gene was amplified using polymerase chain reaction (PCR) and variants of the bST gene were detected as polymorphisms by Alu I restriction endonuclease digestion of PCR products. Restriction enzyme DNA fragments for the leucine127 variant were 265, 96, 51, and 16 base pair and for the valine127 variant were 265, 147, and 16 base pair as a polymorphism of bST was present in the 147 base pair DNA fragment. Frequencies of leucine127 and valine127 alleles for cows (n = 302) were 1.0 and 0 for Brown Swiss, .93 and .07 for Holstein, .92 and .08 for Guernsey, .79 and .21 for Ayrshire, and .56 and .44 for Jersey, respectively. In Holstein sires used for artificial insemination (n = 70), the frequency of leucine127 and valine127 alleles was .96 and .04. Estimates of transmitting ability for milk production tended to be greater for Holstein cows that were homozygous for leucine127 bST and Jersey cows that were homozygous for valine127 bST whereas Holstein sires with different bST genotypes were similar. In summary, frequencies of alleles for the bST gene were not similar in different dairy breeds and estimates of milk production were correlated with bST gene variant in cows but not sires.

A recombinant-DNA-derived modification of human growth hormone (hGH44-191) with enhanced diabetogenic activity.

A modified human growth hormone (hGH) that lacks the first 43 residues of the intact hormone was prepared by recombinant-DNA technology. For preparative purposes an additional alanine was made the amino terminal residue. Sequence analysis and tryptic peptide mapping combined with amino acid analyses confirmed the structure of the polypeptide. Less than 2% N-terminal methionine was detected. The hGH44-191 was estimated to be at least 10 times more active than hGH in producing glucose intolerance in obese yellow mice (Avy/A) and was equipotent to hGH in increasing serum free fatty acids in fasted, hypophysectomized rats. The peptide did not promote growth in hypophysectomized rats nor did it exhibit early (1h) insulin-like activity in fasted, hypophysectomized rats, as indicated by its failure to lower blood glucose and fatty acids. The modified hGH was inactive in the Nb-2 cell assay but was about one-third as active as hGH in stimulating the pigeon crop sac. In radioimmunoassays using 125I-labeled hGH and polyclonal antibodies to intact hGH, cross-reactivity of hGH44-191 was less than 1%. We conclude that removal of the amino terminal portion of hGH enhances its diabetogenic properties, and that this activity does not depend upon the ability to promote growth. Furthermore, the insulin-like activity can be separated from its diabetogenic action by deletion of the first 43 amino terminal residues. This is the first report of a modified hGH that has anti-insulin effects greater than hGH itself.

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.

CHANGES IN INSULIN AND SOMATOMEDIN RECEPTORS AND UPTAKE OF INSULIN, IGF-I AND IGF-II DURING MAMMARY GROWTH, LACTOGENESIS AND LACTATION

Receptors for insulin, IGF-I and IGF-II are present on growing, differentiating and lactating mammary epithelial cells. Scatchard analysis of these receptors indicate the presence of two receptor sites for insulin. In contrast, the type I and type II somatomedin receptors are composed of a single site in all physiological states examined. The type I somatomedin receptor cross-reacted with both insulin and IGF-II while the type II receptor only recognized IGF-II. Although the truncated form of IGF-I has a lower affinity for the type I receptor relative to native IGF-I it is more potent in stimulating mammary growth in vitro. This may be due to mediating effects of binding proteins. Mammary growth appears to be related to higher population of high affinity receptors for IGF-I in mammary tissue during pregnancy. Role of insulin, IGF-I and IGF-II during lactogenesis and galactopoiesis remains to be established.

Introducing strong metal-binding sites onto surfaces of proteins for facile and efficient metal-affinity purifications

Abstract Recombinant DNA methodologies are used to introduce strongly binding, metal-chelating sites onto the surfaces of bovine and porcine somatotropin in such a way that biological activity and protein stability are maintained. Binding to immobilized metals is sufficiently strong and selective that pure variant proteins are efficiently recovered from concentrated or dilute crude cell lysates in one or two purification steps. Chemically robust immobilized metal-affinity matrices are tolerant to repeated use and frequent cleaning and possess high protein-binding capacities. In addition to the preparative uses, immobilized metal-affinity chromatography is a sensitive tool for probing the structures of somatotropin surfaces near metal-binding residues. General principles underlying metal-protein interactions, immobilized metal-affinity chromatography, and variant design are discussed and illustrated.

Generation, characterization and utilization of anti-human growth hormone 1–43, (hGH1–43), monoclonal antibodies in an ELISA

The major isoform of hGH is a polypeptide of 191 amino acids. Human GH1-43 is an amino terminal segment of hGH1-191 which comprises the first 43 amino acids. This peptide is a potent regulator of glucose homeostasis. To facilitate our understanding of the physiological regulation of hGH1-43 an assay to measure its levels in biological fluids and extracts is needed. This communication describes the development of anti-hGH1-43 monoclonal antibodies and their use in the development of an indirect competitive ELISA for the quantification of hGH1-43. Hybridomas were produced by the fusion of FOX-NY myeloma cells with spleen cells taken from a mouse immunized with hGH1-43. The hybridomas were screened for production of antibodies to hGH1-43 by antibody capture ELISA. Hybridomas which produced antibodies reactive to hGH1-43 were cloned by limiting dilution. Three monoclonal hybridomas, CCL-1, CCL-2, and CCL-3 were subsequently obtained. These hybridomas secreted antibodies that were highly reactive towards hGH1-43 but minimally reactive towards hGH1-191. The isotypes of the mAbs secreted by CCL-1, CCL-2 and CCL-3 were all IgG1 kappa as shown by isotype specific antibody capture analysis. An indirect competitive ELISA with a detection limit that ranged from 1 to 10 ng/ml was developed using mAbs from monoclonal hybridoma CCL-3. Dose-response curves for competing hGH1-191, hPRL, and hPL indicated minimal cross-reactivity of mAbs with these hormones and conversely, a high degree of specificity for hGH1-43. Dose-response curves for dilutions of human serum and pituitary extract were parallel to the standard. The availability of a sensitive assay for the measurement of hGH1-43 will help us answer questions regarding the biosynthesis, regulation of secretion, and role of hGH1-43 in the control of glucose homeostasis.

SELECTIVE MODIFICATION OF RECOMBINANT BOVINE PLACENTAL LACTOGEN BY SITE-DIRECTED MUTAGENESIS AT ITS C TERMINUS

Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors.

Synthesis and pharmacological activity of rationally designed inhibitors of the leukotriene A4 hydrolase enzyme

Abstract The synthesis of a series of novel Leukotriene A 4 analogs containing the oxabicycloheptene nucleus has been achieved. These compounds have been evaluated as inhibitors of the Leukotriene A 4 hydrolase enzyme.

Antigenic regions of bovine somatotropin as defined by monoclonal antibodies

Three distinct antigenic regions of bovine somatotropin (bST) were identified on the basis of the ability of a set of monoclonal antibodies to bind to proteolytic fragments and deletion variants of recombinant bST (rbST) in Western blot analyses. One of the regions is further subdivided into two epitopes on the basis of the cross-reaction of somatotropins from several species with the same set of antibodies in solid-phase RIA. The RIA and Western blot results suggest that amino acids 134-150, 181-190 and the amino terminus may be involved in the binding specificity of antibodies to the bovine somatotropin molecule. The total of four antigenic regions on the bST molecule parallels results described for human somatotropin. Labeled antibody competition tests were used to show that the epitope involving amino acids 134-150 is spatially separated from the other three epitopes.