A Moloney MLV-rat somatotropin fusion gene produces biologically active somatotropin in a transgenic pig

Expression of a Moloney murine leukemia virus (MLV) rat somatotropin fusion gene was examined in a transgenic pig. The fusion gene was integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated MLV-rat somatotropin fusion gene produced high levels of circulating rat somatotropin and resulted in an elevation in the circulating levels of insulin-like growth factor I. Although there was no increase in the rate of growth of the transgenic animal during the rapid growth phase, several phenotypic changes were evident. Skeletal growth was markedly increased and fat deposition was reduced throughout the animal. Blood glucose levels were elevated without ketosis. Northern blot analyses of rat somatotropin RNA revealed that expression of the fusion gene was highest in the spleen, lung, intestine, lymph nodes, and bone marrow. These results show that the MLV promoter can be used to express high levels of biologically active rat somatotropin in transgenic swine.     

Hormonal regulation of anabolic processes and of protein utilization efficiency in the early postnatal period

The levels of thyroid, pituitary and steroid hormones-thyroxine, triiodothyronine and 11-hydroxycorticosteroids in the blood serum, somatotropin in the pituitary, and processes of protein assimilation were studied in rats in the early postnatal period. The highest endogenous production of thyroxine, triiodothyronine and somatotropin was detected in 15-day-old rats. The highest level of protein utilization was detected in 7 to 15-day-old rats, followed by the lowering of the utilization on changing over to definitive nutrition. Endogenous production of the anabolic hormones thyroxine, triiodothyronine and somatotropin was found to correlate with a high level of protein utilization in rats within the first days of life after birth.     

基因工程鱼生长激素的生产研究

以P_RP_L,Trp启动子在大肠杆菌中表达鲤鱼生长激素重组DNA,经过高密度发酵.包涵体的提取,恢复天然结构。每升发酵可碍2g鱼生长激素。在上海地区以浸渍方法处理对虾苗并在人工饲料中添加鱼生长激素,提高虾苗的存活率并增加产量,提高了饲料的转化率。     

Modification of bovine somatotropin (growth hormone) with plasmin

Although much work has been reported on modification of human somatotropin with plasmin and has revealed important information about structure-function relationships, plasmin modification of nonprimate somatotropins (which differ markedly in structure and biological actions from the human hormone) has been little studied. Therefore we investigated plasmin digestion of bovine somatotropin. Digestion was less rapid but more extensive than that of human somatotropin. Structural characterization of digestion products resolved by gel filtration suggested that a major product after 24 h was a disulfide-linked fragment comprising residues 1–133 plus 140–177. Further cleavage products were found in aggregated material and minor fractions. In radioimmunoassay for bovine somatotropin, activity was retained only by the unfractionated digest and aggregated material. In radioreceptor assay for somatotropin using receptors from livers of late-pregnant rabbit all preparations retained some activity. The results suggest that receptor- and antibody-binding sites in bovine somatotropin are not identical and that greater activity may result from specific association of fragments that are less active or inactive once separated.     

Dependence of human somatotropin activity on interchain disulfide bridges

The importance of the disulfide bridges for human somatotropin activity is investigated. The activity of somatotropin is tested by the tibia, radioimmuno-, and radioligand assays. The cleavage of disulfide bridges by sulfitolysis, reduction with dithiothreitol, or oxidation with performic acid does not completely abolish hormone activity. There is only one exception: in the radioligand assay, oxidized somatotropin is not able to displace native somatotropin from rat liver membranes. The diminution of hormone activity is independent of the charges of the groups introduced to the cysteine residues. The radioimmuno- and radioligand assays are more sensitive to conformational alterations in the somatotropin molecule than the biological test system.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Purification and characterization of pituitary bovine somatotropin

Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli. Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method. NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences. The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected. Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent. Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128.     

Formation of complexes between 125I-labelled human or bovine somatotropins and binding proteins in vivo in rat liver and kidney.

At 5 min after intravenous injection, both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin were concentrated in rat liver and kidney. When the labelled hormones were administered along with an excess of the corresponding unlabelled hormone, a significant decrease of the uptake was observed in the liver, but not in the kidney. Study of the subcellular distribution of radioiodinated somatotropins in liver revealed that most of the radioactivity was specifically concentrated in the microsomal fraction. In contrast, the kidney fraction that accounted for most of the radioactivity was the 100 000 g supernatant. After solubilization, with 1% (w/v) Triton X-100, of the microsomal fractions obtained from both organs, the radioactive material was analysed by gel filtration on Sepharose CL-6B. By using this approach, it was demonstrated that both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin bind in vivo to proteins present in liver. A small proportion of 125I-labelled human somatotropin was also shown to form complexes with proteins present in kidney. The present results demonstrate that the liver uptake is mainly due to binding of somatotropins to specific proteins, in contrast with the kidney, in which binding to specific sites contributes minimally to the overall uptake.     

Human somatotropin binding to rabbit kidney microsomal fraction.

Specific binding of 125I-labelled human somatotropin was demonstrated in microsomal membranes (microsomes) from rat and rabbit kidneys. Female rabbit kidney microsomes showed the highest binding activity and were used for further study. The association of 125I-labelled human somatotropin was time- and temperature-dependent and the binding reaction was reversible. Scatchard analysis of saturation data indicated a dissociation equilibrium constant, KD, of 56 pM and a binding capacity of 37 fmol per mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the microsomes was specifically inhibited by hormones with lactogenic activity. The binding sites, as well as 125I-labelled human somatotropin, were not inactivated on incubation. Treatment of the microsomes with trypsin and chymotrypsin decreased the specific binding by over 90%. Preheating of the microsomes at 55 degrees C for 15 min abolished 50% of the specific binding activity.     

A novel concatenated dimer of recombinant bovine somatotropin

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized inEscherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.     

Primary structure of cyanogen bromide fragments of sei whale (Balaenoptera borealis) pituitary somatotropin]

5 fragments are isolated after the degradation of somatotropin from sei whale pituitary glands with cyanogen bromide: N-terminal 4-segmented; C-terminal 12-segmented with the internal disulfide bond; middle 25- and 30-segmented and a high molecular weight fragment following N-terminal tetrapeptide and bound with disulfide bond to 30-segmented fragment. Complete amino acid sequence of three shortest cyanogen bromide fragments is deciphered and N- and C-terminal sequence is investigated in two large fragments after their uncoupling under performic acid oxidation. Amino acid sequence is deciphered of a peptide obtained after trypsine hydrolysis of 30-segmented cyanogen bromide fragment. Comparison of amino acid sequence of whale somatotropin fragments with that of sheep, beef and human somatotropin has revealed that 57 out of 61 identified amino acid residues of whale somatotropin repeat amino acid residues in similar regions of beef somatotropin, 56--of sheep and only 42--of human somatotropins. Besdies, 4 of 5 revealed amino acid substitutions in whale hormone, as compared with sheep somatotropin, are amino acids which are present at the same positions in human hormone.     

Analysis of therapeutic growth hormone preparations: report of an interlaboratory collaborative study on growth hormone assay methodologies.

Recombinant DNA-derived human growth hormone (somatotropin) is widely used to treat growth hormone-deficient children. The potency of this product is determined by in-vivo bioassay in hypophysectomized rats, which is imprecise, costly and invasive, and there have been suggestions that it could safely be replaced with in-vitro or physico-chemical alternatives. In this report we present the results of a collaborative study designed to test this proposal. Somatotropin was modified by mild or severe proteolysis, mild or severe oxidation or treatment at high pH, and compared in a multi-centre collaborative study with unmodified somatotropin or with dimerized somatotropin. Participating laboratories included manufacturers and national control laboratories, and pharmacopoeial bioassays were compared with in-house in-vitro and physico-chemical bioassays. Although performing adequately with untreated somatotropin, for degraded samples the in-vivo bioassays were relatively unresponsive to changes in the growth hormone molecule. In contrast, the physico-chemical assays, in particular the reverse-phase HPLC, performed with a high degree of selectivity. We conclude that in the case of somatotropin, the in-vivo bioassay can be removed from the routine product specification with an acceptable degree of security. This however does not obviate the requirement rigorously to demonstrate biological activity in-vivo during product development, nor may the conclusions of this study be applied to other therapeutic recombinant proteins without similar collaborative investigations.     

Preparation of internally labelled rat pituitary somatotropin (growth hormone).

Rat somatotropin (growth hormone) was labelled biosynthetically by incubating anterior pituitary lobes with radioactive amino acids for 24 h in a simple buffered salts medium containing glucose. The labelled hormone was isolated by preparative polyacrylamide-gel electrophoresis or by chromatography on Sephadex G-100 and then DEAE-cellulose. The labelled material was pure by several criteria and cross-reacted immunologically with unlabelled rat somatotropin. When a mixture of 14C-labelled amino acids was used for labelling the protein, label could be introduced into these same amino acids of somatotropin, though relative specific radioactivities varied considerably. Somatotropin labelled by the procedures described in the present paper was suitable for structural studies and could be used for a variety of other biochemical experiments.     

Thermogenic and lipogenic activities in brown adipose tissue of I-strain mice.

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.     

Maternal treatment with somatotropin during early gestation affects basic events of myogenesis in pigs

The effects of maternal treatment with somatotropin during early gestation on fetal muscle development were determined. Crossbred gilts received daily injections of either 3 ml of a placebo ( n=31) or of 6 mg porcine somatotropin ( n=31) from day (d) 10 to 27 of gestation and samples were collected from d 28 embryos, d 37 and 62 fetuses, and from neonates. Administration of somatotropin increased the total number of fibres (primary and secondary fibres) in neonatal semitendinosus muscle of middle- and low-weight littermates, whilst no increase was observed in psoas major muscle. Somatotropin induced increases in muscular protein concentration, creatine kinase activity, muscle fibre girth, as well as type II to type I fibre conversion which revealed an advanced degree of differentiation at birth. Treatment effects on prenatal development preceded these changes. Increased DNA concentrations at d 28 of gestation indicate stimulation of cellular proliferation during the embryonic stages. Thereafter, the withdrawal of somatotropin caused a transient delay of differentiation as indicated by lower protein concentrations and creatine kinase activity compared with controls at d 37 of gestation. This was compensated again at d 62, and the number of semitendinosus primary fibres was increased in middle-weight fetuses, whereas secondary or total fibre number did not yet differ. However, enhanced expression of Myf5 and MyoD indicates higher numbers of initially determined, proliferating myoblasts that may have contributed to increased formation of secondary fibres. In conclusion, maternal somatotropin is an influential factor in early pregnancy capable of affecting the basic events of myogenesis.     

Comparative analytical studies on recombinant and native human somatotropin

In polyacrylamide gel electrophoresis and isoelectric focusing, somatotropin produced by recombinant DNA technology is as heterogeneous as highly purified native pituitary somatotropin. This heterogeneity is not attributable to different degrees of deamination of a single molecular species. In addition to the main protein of 22 kDa, the cloned products contain traces of interchain disulphide dimers of somatotropin. The quantitative amino acid analyses of the three cloned somatotropins investigated are neither identical nor do they correspond to the analysis of the native pituitary hormone. Moreover, there are discrepancies between the amino acid compositions of the hormones studied and the generally recognized amino acid analysis for human somatotropin.     

The influence of somatotropin, prolactin and insulin on granulosa cells from bovine atretic follicles

An influence of somatotropin, prolactin and insulin on destructive processes in bovine granulosa cells from small antral follicles following atresia in vivo was studied in vitro. As compared to control, the addition of the studied hormones to serum-free suspension system was shown to result in increase in number of cells without signs of chromosome degeneration after 24 and 48 hrs of incubation. The revealed inhibitory action of somatotropin, prolactin and insulin on chromatin degeneration in granulosa cells was not due to the hormonal influence on proliferative activity of the cells. The stimulatory action of insulin on the viability and estrogen-secretory activity of granulosa cells cultured for 1 day was also found. At the same time, somatotropin and prolactin did not affect the estradiol and progesterone production by the cells. The data obtained suggest that the inhibitory action of somatotropin and prolactin on destructive processes in cultured granulosa cells is not related to the hormonal regulation of the steroidogenic activity of the cells, whereas the similar action of insulin may be partially due to its stimulatory influence on the estradiol secretion.     

Changes of plasma insulin, urea, amino acids and rumen metabolites in somatotropin treated dairy cows

Summary An experiment was performed to evaluate the effects of somatotropin on plasma free amino acid, urea and insulin concentrations and rumen fermentation pattern and to assess their relationships. Four Italian Friesian dairy cows fitted with rumen cannulae were used in a switch-back design. Slow releasing recombinant bovine somatotropin (640 mg/cow) was injected every 28 days for two consecutive periods. Rumen fluid and blood samples were collected before and after feeding at 0, 7 and 21 days after rbST injection. Exogenous rbST increased plasma insulin concentration and the insulin response to feeding, and decreased plasma urea and free essential and branched chain amino acid concentrations. rbST did not affect rumen fermentation pattern. No correlation was found between rumen and plasma parameters measured after feeding. Our results are consistent with the notion that the main effect of somatotropin is post-absorptive.     

Preparation of highly purified human somatotropin (growth hormone)

A method for preparation of highly purified human somatotropin on large-scale basis is described. Starting from deep-frozen pituitary gland, less time is needed to obtain highly purified hormone than with other published methods for preparation of human somatotropin. The hormone obtained in this fasion is chromatographically and electrophoretically homogeneous; it shows high biological and radioimmunological growth hormone activity and is free of other pituitary hormone activities. The effects of various experimental conditions upon aggregation of somatotropin are critically evaluated.