Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways.

OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.

Expression and complement d activity of porcine adipsin.

To learn how signals from adipocytes might be involved in regulation of energy intake and storage, we have begun to characterize the porcine complement protein, adipsin. Adipsin was originally identified as a protein that is produced rather specifically by adipocytes, is secreted, and is nearly absent in several obese rodent models. We now report that porcine adipsin mRNA sequence is 74% identical to rat and predicts a protein that has 82 and 68% identity to human and rat forms, respectively. Porcine adipsin has none of the asparagine glycosylation consensus sites which make recombinant expression of mouse adipsin in Escherichia coli impractical. We present a method for engineering the porcine cDNA to facilitate expression by E. coli and provide a protocol for refolding and purifying porcine adipsin protein and for immunoassay. We have found that in addition to adipose tissue, adipsin mRNA is present in gut tissues. Coupled with the fact that adipsin is required for processing of complement C3a-desArg, and that C3a-desArg is a potent stimulant of fatty acid acylation in adipocytes, the production of adipsin in the gut may be related to a mechanism for adipocyte removal of lipid from chylomicrons.

Evaluation of the somatogenic activity of bovine placental lactogen with cell lines transfected with the bovine somatotropin receptor.

Studies have shown that bovine placental lactogen (bPL) has partial somatogenic activity in vivo even though binding results clearly indicate bPL does not cause homodimerization of the bovine somatotropin receptor (bST-R). To help understand the receptor binding versus biological activity of bovine somatotropin (bST) and bPL we have developed a homologous model system. Full length bST-R was stably transfected into a murine lymphoid cell line, Ba/F3 and a hamster kidney cell line, BHK. From both transfected cell lines, clones were isolated (Ba/F3-C1 and BHK-24) which demonstrated specific binding of bST and, or bPL. Bovine ST stimulated proliferation of the Ba/F3-C1 clonal line over a dose range of 10 to 3000 pM with an EC50 of 100 pM. A bST variant (des 1-4 bST) and porcine ST (pST) which both have approximately 10% of the binding affinity for bST-R as native bST were 1 and 10% as potent as bST in this bioassay, respectively. This suggests that affinity and biological activity are correlated for this system. Proliferation was initiated through the bST-R because addition of a monoclonal antibody which recognizes the extracellular domain of bST-R and inhibits binding of bST to its receptor, inhibited bST-stimulated mitosis. However, even though the affinity of bPL for the bST-R is similar to that of bST, bPL antagonized the proliferative action of bST with an IC50 of 1 nM. Components of the somatogenic signal transduction pathway were also evaluated in both cell lines. Addition of bST to the cell cultures increased phosphorylation of JAK2 in Ba/F3-C1 and BHK-24 cells in a dose-responsive manner but bPL failed to increase phosphorylation of JAK2 in either cell line. In summary, these data support the hypothesis that ST-R homodimerization is necessary for bioactivity in this model system but fail to explain apparent somatogenic activity of bPL in vivo.

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.

Novel recombinant analogues of bovine placental lactogen. G133K and G133R provide a tool to understand the difference between the action of prolactin and growth hormone receptors.

Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form. Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity. The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration. Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested. Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR- and GHR-ECDs. The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished. Both analogues exhibited antagonistic activity in the latter cells. To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization.

Site-directed mutagenesis of recombinant bovine placental lactogen at lysine-73 leads to selective attenuation of its somatogenic activity.

Bovine placental lactogen (bPL) is capable of binding and transducing biological activity via somatogenic and lactogenic receptors. To modify this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), were produced in Escherichia coli. Circular dichroic spectrum analyses indicated proper refolding in all cases. Biological activity of these analogs was tested in vitro. In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly. In another lactogenic bioassay that measures beta-casein synthesis by HC-11 mouse mammary-gland cells, the analogs were 30-40% as potent as bPL. In contrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells transfected with either rabbit (rb) or hGH receptor (R) was almost completely abolished in these analogs. In receptor binding assays, the effect was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding. Binding to rat (r) and rabbit PRL receptor extracellular domains (ECDs) or membrane-embedded receptors was only slightly changed, except for bPL (K73D), which displayed very low affinity. In somatogenic binding assays to intact IM-9 human lymphocytes, hGHR-ECD or bovine liver membranes, bPL (K73D) did not bind at all, and bPL(K73F) or bPL(K73A) binding was drastically reduced. Binding experiments performed in real time using a BIAcore apparatus revealed that the decreased binding could be mainly attributed to increased k(off) rather than decreased k(on) values. The complex with hGHR-ECD revealed a 2:1 stoichiometry with bPL, bPL(K73F) and bPL(K73A), although the complex with these analogs was less stable than with bPL, whereas bPL(K73D) scarcely assembled a 1:1 complex. In contrast, bPL and the three analogs formed stable 1:2 complexes with rPRL-ECD. These results suggest that position 73 in bPL is more important for somatogenic than lactogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same differential importance with respect to somatogenic binding.

Human growth hormone deletion mutant (hGH44-191) binds with high affinity to lactogenic receptors but not to somatogenic receptors.

The carboxyl-terminal hGH fragment, hGH44-191, displays diabetogenic activity. To understand whether this biological activity is mediated through somatogenic or lactogenic receptors, we investigated the ability of hGH44-191 to bind both receptor classes. We found that hGH44-191 could not compete with [125I]hGH or [125I]bGH for bovine liver somatogenic binding sites. Additionally, hGH44-191 could not displace [125I]hGH from the somatogenic receptor sites when human liver microsomes were used as the receptor source. In contrast, hGH44-191 effectively competed with [125I]hGH for lactogenic receptor sites of lactating mammary gland microsomes and of bovine liver microsomes. In summary, hGH44-191 does not bind to somatogenic receptors but does not bind to lactogenic receptors. These data suggest that the biological actions of hGH44-191 could be mediated through lactogenic receptors.

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.

N-terminal-truncated recombinant analogs of bovine placental lactogen: interaction with human and rat growth hormone receptors and insulin-like growth factor-I secretion mediated by somatogenic receptors in rat hepatocytes.

Bovine placental lactogen (bPL) was found to be as potent as human GH (hGH) in its ability to bind to soluble full-size recombinant hGH-binding protein (hGHBP) and to membrane-embedded hGH receptor in intact IM-9 human lymphocytes. bPL was also capable of forming a 1:2 complex with hGHBP, although the structure of this complex was probably more compact than that with hGH. Removal of 13 amino acids from the N-terminus of bPL did not affect its ability to bind to hGHBP or hGH receptors in intact IM-9 cells. Its ability to form a 1:2 complex with hGHBP was, however, impaired, unlike that of a corresponding analog in which an L28F mutation has been simultaneously introduced. Truncation of 17 amino acids decreased its affinity toward both hGHBP and GH receptors on intact IM-9 lymphocytes and in liver rat microsomal fraction and inhibited the formation of 1:2 complexes with hGHBP. Simultaneous L28F mutation did not affect affinity toward hGHBP, but increased affinity toward rat liver GH receptors and restored affinity toward membrane-embedded hGH receptors in IM-9 lymphocytes and the ability to form a 1:2 complex with hGHBP. Truncation of 20 amino acids further decreased affinity toward both hGHBP and receptors in intact IM-9 lymphocytes and completely abolished formation of a 1:2 complex with hGHBP. Both des-13-bPLs and bPL-des-17 (L28F) retained their full ability to stimulate insulin-like growth factor-I secretion by rat hepatocytes compared to that of bPL. The insulin-like growth factor-I stimulatory activities of bPL-des-17 and bPL-des-20, however, were decreased to 1-5%. These results indicate that the stoichiometry of 1:2 complex formation with hGHBP may be preserved despite decreased receptor binding affinity, but the lower affinity of the putative site 1 or site 2 of the analog may account for the decrease in biological activity. Furthermore, the ability or inability of bPL or its truncated analogs to form 1:2 complexes with soluble hGHBP cannot predict their somatogen receptor-mediated biological activity in rat hepatocytes.

Extracellular domain of prolactin receptor from bovine mammary gland: expression in Escherichia coli, purification and characterization of its interaction with lactogenic hormones.

The cDNA of the extracellular domain of bovine prolactin receptor (bPRLR-ECD) was cloned and expressed at high yield as an insoluble protein in Escherichia coli. This protein was solubilized, refolded and purified to > 98% homogeneity yielding 80 mg of monomeric fraction per 2 litres of induced culture. Its molecular mass was 25.7 kDa, as determined by SDS-PAGE in the absence of reducing agent and 24 kDa by gel filtration on a Superdex column. Binding experiments revealed that bPRLR-ECD binds to human (h) GH (hGH) with high affinity, whereas its affinity for ovine (o) or bovine (b) prolactins (PRLs) was lower and for bovine placental lactogen (bPL) very low. The affinity of bPRLR-ECD for the latter three hormones was, however, much higher than that of membrane-embedded or solubilized bPRLR. The stoichiometries of interaction of bPRLR-ECD with hGH, oPRL, bPRL and bPL were determined by gel-filtration chromatography. Even at a 3:1 ECD excess, only 1:1 complexes were detected at microM concentrations of ECD and ligand. At an up to 32-fold dilution, the complexes with oPRL, bPRL, and particularly bPL, underwent progressive dissociation, whereas the complex with hGH remained stable. Although all four hormones exhibited nearly identical activity in the Nb2 lymphoma cell bioassay, the ability of bPRLR-ECD to inhibit hormonal mitogenic activities differed, generally reflecting its affinity for the respective hormones. In view of these and previous results, we suggest that, unlike in the GH:GHR-ECD interaction, neither the stoichiometry of interaction of bovine or other PRLR-ECDs nor the affinity constants can predict the biological potency of the different lactogenic hormones.

Interaction of lactogenic hormones with the soluble extracellular domain of prolactin receptors.

Two variants of rabbit prolactin receptor extracellular domain (rbPRLR-ECD) were prepared using insect/baculovirus (amino acids 1-198) and E. coli (amino acids 4-210) expression systems. Bovine PRLR-ECD (bPRPL-ECD amino acids 1-210) and human growth hormone receptor ECD (hGHR-ECD amino acids 1-246) were also prepared using E. coli expression system. All four proteins were purified as monomers with > 95% homogeneity. Their affinity for various lactogenic and somatogenic hormones was determined by binding assays. The stoichiometry of complex formation with these hormones was studied by gel filtration on a Superdex 75 column, and bioactivity was determined by in vitro bioassays. The results summarized in this paper indicate that, in contrast to hGHR-ECD, in which the ability to form a 2:1 complex with hGH is indicative of the biological activity of the hormone, the ability or inability of prolactin and placental lactogen to form 2:1 complexes with rb or bPRLR-ECD cannot predict their biological activity. This conclusion does not preclude however, hormone- or antibody-induced dimerization of the membrane-embedded receptor.

Ligand-specific dimerization of the extracellular domain of the bovine growth hormone receptor.

The bovine growth hormone (bGH) receptor and its extracellular domain (bGHBP) bind two protein hormones with high affinity; bGH and bovine placental lactogen (bPL). However, each of these hormones bind with a different stoichiometry. bGH binds to the bGHBP in a 1:2 ratio while bPL binds in a 1:1 ratio. Scatchard analysis of saturation binding yields similar apparent dissociation constants (Kd) for bGH and bPL of 1.4 x 10(-11) M and 3.0 x 10(-11) M, respectively, for the membrane receptor and 6.8 x 10(-11) M and 4.2 x 10(-11) M, respectively, for bGHBP. In competition experiments using either liver membranes or bGHBP, bGH is 2-3-fold more effective than bPL in competing for 125I-bGH-binding sites. In similar experiments using 125I-bPL, bPL is 50-fold more effective than bGH in competing for binding sites. A rat monoclonal antibody raised against bGHBP competes effectively for 125I-bGH-binding sites, but not for 125I-bPL-binding sites. Since bPL cannot dimerize the bGHBP and yet it acts in part as a somatogen in vivo, homodimerization of the growth hormone receptor is apparently not essential for some biological responses signaled through this receptor.

Stimulation of food intake and weight gain in mature female rats by bovine prolactin and bovine growth hormone.

Recombinant bovine prolactin (rbPRL) or bovine growth hormone (rbGH) was administered to mature female rats (10/treatment group) by daily subcutaneous injection for 10 days. Doses ranged from 7 to 5,000 micrograms/day (0.03-24 mg/kg body wt). Both rbPRL and rbGH increased body weight gain and food intake, but these parameters were increased at lower doses of rbPRL (7-63 micrograms/day) than rbGH (> 190 micrograms/day). Weight gain and food intake were maximally stimulated by 190 micrograms/day rbPRL, whereas maximal increased weight gain was obtained with the highest dose of rbGH (5,000 micrograms/day). Total carcass protein was increased by both hormones; however, protein as a percentage of body weight was unchanged. Similarly, neither rbPRL nor rbGH changed the percentage of carcass moisture. Percentage of body fat was increased by rbPRL but was decreased by rbGH. Weight of the gastrointestinal tract and kidneys was increased by both hormones, but increases were in proportion to body weight gain. These data confirm that ungulate prolactin is a hyperphagic agent in the female rat. In addition, they suggest that, while prolactin stimulates growth in mature female rats, this growth is probably not via a somatogenic mechanism.

Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.

Ruminant placental lactogens: structure and biology.

Ruminant placental lactogens (PL) are members of the somatotropin, prolactin gene family that are synthesized by trophectodermal binucleate cells. The structure and biology of PL has been studied in the cow, sheep, and goat. Ruminant PL have greater structural identity to prolactin than somatotropin, although they bind to both lactogenic and somatogenic receptors. The molecular weights of ovine and caprine PL are approximately 23,000, whereas bovine PL is larger (31,000 to 34,000) due to glycosylation. Placental lactogen is secreted into both the fetal and maternal circulations. The concentration of PL in the fetus decreases with advancing gestation, whereas PL concentration peaks in the maternal circulation during the last third of pregnancy then reaches a plateau. Furthermore, the maternal concentration of PL is 100- to 1,000-fold higher in sheep and goats than in cows. The precise factors that modulate secretion of PL are unknown, although placental mass and nutrition seem to play a role. Ruminant PL have both lactogenic and somatogenic biological activities and may also have unique activities mediated through a specific receptor. There is circumstantial evidence to suggest that PL plays a role in stimulating mammogenesis. Placental lactogen secreted into the fetal compartment may also help regulate fetal growth. Direct experimental data indicate that PL can regulate maternal intermediary metabolism. Thus, it may act as a partitioning agent to regulate nutrient supply for fetal growth. The precise biological function of PL in ruminants, therefore, still needs to be defined.

Enhanced heterologous gene expression in novel rpoH mutants of Escherichia coli.

Extragenic temperature-resistant suppressor mutants of an rpoD800 derivative of Escherichia coli W3110 were selected at 43.5 degrees C. Two of the mutants were shown to have a phenotype of enhanced accumulation of heterologous proteins. Genetic mapping of the two mutants showed that the mutation conferring temperature resistance resided in the rpoH gene. P1-mediated transduction of the rpoD+ gene into both of the rpoD800 rpoH double mutants resulted in viable rpoH mutants, MON102 and MON105, that retained temperature resistance at 46 degrees C, the maximum growth temperature of W3110. The complete rpoH gene, including the regulatory region, from MON102, MON105, and the parental W3110 was cloned and sequenced. Sequencing results showed that a single C----T transition at nucleotide 802 was present in both MON102 and MON105, resulting in an Arg(CGC)----Cys(TGC) substitution at amino acid residue 268 (R-268-C; this gene was designated rpoH358). Heterologous protein accumulation levels in both MON102 and MON105, as well as in rpoH358 mutants constructed in previously unmanipulated W3110 and JM101, were assessed and compared with parental W3110 and JM101 levels. Expression studies utilizing the recA or araBAD promoter and the phage T7 gene 10L ribosome-binding site (g10L) showed that increased accumulation levels of a number of representative heterologous proteins (i.e., human or bovine insulin-like growth factor-1, bovine insulin-like growth factor-2, prohormone of human atrial natriuretic factor, bovine placental lactogen, and/or bovine prolactin) were obtained in the rpoH358 mutants compared with the levels in the parental W3110 and JM101. The mechanism of enhanced heterologous protein accumulation in MON102 and MON105 was unique compared with those of previously described rpoH mutants. Pulse-chase and Northern (RNA) blot analyses showed that the enhanced accumulation of heterologous proteins was not due to decreased proteolysis but was instead due to increased levels of the respective heterologous mRNAs accompanied by increased synthesis of the respective heterologous proteins. The plasmid copy number remained unaltered.

Stimulation of body weight gain of the mature female rat by bovine GH and bovine placental lactogen.

Mature female rats (200 g) were treated for 10 days with either recombinant bovine GH (bGH) or recombinant bovine placental lactogen (bPL) to compare the somatogenic responses elicited by these hormones. The treatments were administered by daily s.c. injection at four dose levels (0.19, 0.56, 1.67 and 5.0 mg/day). Both bGH and bPL stimulated significant increases in weight gain, but the slopes of the dose-response curves were different (P less than 0.05). Bovine PL was more potent than bGH (P less than 0.01) at the lowest dose, although there were no differences between treatment groups at the three higher doses. Feed consumption was stimulated more by bPL than bGH at all doses (P less than 0.001). The concentration of insulin-like growth factor-I (IGF-I) in blood plasma was increased by bGH in a dose-responsive manner and was higher than control at doses of 1.67 and 5 mg/day (P less than 0.05). Low doses of bPL stimulated increases in IGF-I similar to those with bGH. At the highest dose of bPL, however, there was no concomitant increase in plasma IGF-I. Nevertheless, the growth rate of the animals in this group matched that of the group given the highest dose of bGH. Receptor binding studies indicated that bPL bound to both GH and prolactin receptors. This is consistent with the growth data which suggests that bPL stimulated weight gain through a somatogenic mechanism as well as by another route, possibly mediated by lactogenic receptors.

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.