Metabolic effects of plasmin digests of human growth hormone in the rat and man.

As a first step in our study of structure-function relationships among primate and non-primate growth hormones, human growth hormone (hGH) was subjected to the limited digestive activity of human plasmin. The lyophilized whole digest, containing less than 2% of unchanged hormone, had an average of 2.3 new amino-terminal groups per mole. The digest had the same potency as the native hormone (a) in causing weight gain in hypophysectomized rats; (b) in stimulating somatomedin production in hypophysectomized rats; (c) in stimulating upake of [(3)H]leucine into isolated diaphragm of hypophysectomized rats; (d) in accelerating transport of [(14)C]alpha-aminoisobutyric acid into isolated diaphragm of hypophysectomized rats; (e) in stimulating uptake of [3-0-methyl-(14)C]glucose by isolated adipose tissue of hypophysectomized rats; (f) in accelerating conversion of [(14)C]glucose to (14)CO(2) by isolated epididymal adipose tissue of hypophysectomized rats. The digest also caused glucosuria in partially pancreatectomized rats treated with dexamethasone. These metabolic actions of plasmin-digested hGH in the array of animal tests were confirmed by comparable effects elicited in 11 human subjects (nine pituitary-deficient children and adolescents and two nondeficient adults). A single injection of the plasmin digest caused an increase in plasma free fatty acids and a fall in plasma amino acids. Seven daily injections caused positive balances of nitrogen, phosphorous, sodium, and potassium, gain in body weight, and in two of three subjects impairment of glucose tolerance. The potency of the plasmin digest in producing these metabolic effects in man was comparable to that of native hGH.Thus, 2-3 bonds in the hGH molecule can be cleaved by plasmin without impairing the hormone's growthpromoting, anabolic, diabetogenic, and adipokinetic actions for rat and man.

Possible evidence for the existence of a growth hormone fragment in porcine pituitary.

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126-180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of 125I-labelled fragment B by procine growth hormone required a 10(3) M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of 125I-labelled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possible representing a growth hormone fragment.

Correlation between the biological and immunological activities of growth hormone circulating in rats bearing MtTW15 tumors.

Growth hormone-like activity was demonstrated in diluted plasma of rats bearing the GH-secreting tumor, MtTW15, using an in vitro bioassay. The bioassay used depends upon the ability of GH in vitro to stimulate the uptake of 3-0-methyl glucose (3-OMG) into the isolated diaphragm of the hypophysectomized rat. It was found that the effect of the diluted plasma on this system was qualitatively like that of rat pituitary GH. Theophylline, a drug which blocks the action of GH on 3-OMG uptake by the diaphragm but has no effect on the actions of insulin or somatomedin on this process, completely abolished the stimulatory action of the plasma on the transport of the sugar. Furthermore, antibodies against rat pituitary GH (ArGH) completely abolished the biological activity of the diluted plasma. When plasma was diluted to give specific concentrations of rat GH (rGH), as determined by radioimmunoassay (RIA), the plasma produced responses in the 3-OMG assay equivalent to those obtained with similar concentrations of pituitary rGH. A good correlation was also observed between the concentration of rGH in the plasma of tumor-bearing rats measured by RIA and by an in vivo bioassay in which the effects were determined of injections of diluted plasma into hypophysectomized rats on the subsequent incorporation of [3H]thymidine into costal cartilage. This in vivo biological activity was abolished by incubation of the plasma with ArGH prior to its injection into the test animals. Thus, these studies indicate that in rats bearing the MtTW15 tumor there is a good correlation between the biological and immunological activities of the rGH circulating the bloodstream.

Isolation and biological characterization of fragments of human growth hormone produced by digestion with plasmin.

The biologically active component of plasmin digested human growth hormone consisted of residues 1-134 attached to residues 141-191 by the disulfide bond between residues 53 and 165. This large fragment of the hormone retained the in vivo capacity to stimulate weight gain and cartilage metabolism in hypophysectomized rats and exhibited the diabetogenic property of the native hormone in partially pancreatectomized, dexamethasone-treated rats. This fragment also retained the in vitro ability to stimulate protein synthesis and amino acid and sugar transport into isolated diaphragm muscle of hypophysectomized rats, Furthermore, a smaller peptide (residues 1-134) derived from the large fragment by reduction and carbamidomethylation of the disulfide bond retained high activity in in vitro systems and the in vivo capacity to stimulate cartilage metabolism provided the peptide was injected intravenously. Thus the present studies demonstrate that many of the in vivo and in vitro metabolic effects of human growth hormone on tissues of hypophysectomized rats can be elicited with a peptide comprising the aminoterminal 134 amino acid residues of native human growth hormone.

Isolation and characterization of fragments of reduced and S-carbamidomethylated human growth hormone produced by plasmin digestion. I. Chemistry.

Reduced and S-carbamidomethylated human GH (RCAM-hGH) was digested with human plasmin, yielding a mixture of products. These were partially separated by chromatography on DEAE-cellulose, yielding three major fractions: Da and Db, which were equipotent with native hGH in the weight gain test; and Dc, which was about half as active as hGH. Each of these was further purified by gel filtration, yielding a number of subfractions which were characterized as follows: Da1 is a very stable noncovalent complex of residues 1--134 and 141--191 of RCAM-hGH; Da2 represents residues 20--41; Db1 is very similar to Da1, but appears to have lost one or more amide groups; Db3 represents residues 95--134; Dc2 is a heterogenous fraction containing a further deamidated version of Da1 and Db1 plus a similar complex of residues 42--134 and 141--191, apparently with some carboxyterminal heterogeneity; Dc3, like Db3, represents residues 95--134. The biological activities of these fragments are discussed in the accompanying paper. Earlier work has shown that native hGH, upon digestion with plasmin, is cleaved primarily at residues 134 and 140. It is shown here that when RCAM-hGH is digested with plasmin, in about 87% of the molecules at least one cleavage takes place in addition to those at residues 134 and 140.

Ten years of basic medical physiology in the Mercer problem-based curriculum.

In our curriculum, students learn basic medical physiology and pathophysiology during a 74-week integrated multidisciplinary program. This problem-based program consists of two phases aimed at student acclimation to the educational approach and to coverage of fundamental information, followed by 10 phases devoted to in-depth coverage of the organ systems. Physiological principles are given major emphasis during these latter 10 phases. In this approach, students meet in small groups, identify basic science learning issues (including physiology) from written biomedical cases, research the issues, and discuss these issues in relation to each case. These groups (6-7 students plus a tutor) meet for 3-hour sessions three times each week during each phase. Students receive cases, along with study guides designed to assist in selecting appropriate information sources for each phase. Each student is evaluated on group process skills, oral presentation and defense of a case analysis, and a multiple-choice exam. Internal and external [National Board of Medical Examiners (NBME) Part I] evaluations for the Classes of 1987-1993 indicate that our problem-based approach results in student learning of medical physiology.

Protein synthesis inhibitors and gastric acid secretion.

The involvement of de novo protein synthesis in acid secretion by the in vitro bullfrog gastric mucosa was examined using the inhibitors cycloheximide and puromycin. Both inhibitors reduced [3H]leucine incorporation by more than 90%. The inhibition of protein synthesis had no influence on spontaneous acid secretion nor did it effect secretory stimulation by histamine, carbachol, pentagastrin, or theophylline. Oxygen consumption by nonstimulated and theophyllinestimulated tissues was shown to be independent of protein synthesis. The dramatic morphological changes observed in oxyntic cells during a transition from rest to active secretion persisted in cycloheximide-and puromycin-treated tissues. Stereological analysis of the apical membrane surface area density failed to demonstrate any quantitative influence of protein synthesis inhibition. These studies quantitatively confirm that protein synthesis is not required for the biochemical and morphological events involved in acid secretion by the in vitro frog gastric mucosa.

Biological properties of plasmin digests of S-carbamidomethylated human growth hormone.

Reduction and carbamidomethylation of the intrachain disulfide bridges of human growth hormone did not destroy its ability to stimulate weight gain or cartilage metabolism in hypophysectomized rats. The reduced and alkylated hormone also stimulated glucose oxidation in isolated adipose tissue of hypophysectomized rats when added in vitro. When the S-carbamidomethylated hormone was incubated overnight with human plasmin, approximately 95% of the starting material was completely digested, as judged by polyacrylamide gel electrophoresis. The plasmin digest retained the ability to stimulate weight gain, cartilage metabolism, and glucose oxidation. A fraction consisting of two major electrophoretic components was isolated from the digest by chromatography on tsephadex G-50. This fraction possessed the biological properties of the whole digest.

Bioactive factors affect proliferation and phenotypic expression in progenitor and pluripotent stem cells.

Progenitor and pluripotent stem cells reside within connective tissue compartments. They are also present in granulation tissue. This study examined the effects of treating these two cell populations with eight bioactive factors. Cells were assayed for DNA content as a measure of proliferation and for tissue-specific phenotypic markers as measures of lineage progression and lineage commitment. Platelet-derived endothelial growth factor and insulin-like growth factor-II did not induce proliferation in either population. However, dexamethasone, insulin, insulin-like growth factor-I, muscle morphogenetic protein, platelet-derived growth factor-AA, and platelet-derived growth factor-BB stimulated proliferation in one or both cell populations. Platelet-derived growth factor-BB was the most potent stimulator of proliferation in either population. Phenotypic expression markers were induced in the progenitor cells by insulin, insulin-like growth factor-I, insulin-like growth factor-II, dexamethasone, and muscle morphogenetic protein. However, only dexamethasone and muscle morphogenetic protein induced phenotypic expression markers in the pluripotent cells. Platelet-derived endothelial cell growth factor, platelet-derived growth factor-AA, and platelet-derived growth factor-BB did not induce phenotypic expression markers in progenitor or pluripotent cells. This study suggests the potential for using progenitor and pluripotent cells as an in vitro model to ascertain the effects of various bioactive factors on stem cells potentially involved in tissue maintenance and repair.

Mesenchymal stem cells reside within the connective tissues of many organs.

Previous studies have noted the presence of mesenchymal stem cells located within the connective tissue matrices of avian skeletal muscle, dermis, and heart. In these studies, clonal analysis coupled with dexamethasone treatment revealed the presence of multiple populations of stem cells composed of both lineage-committed progenitor mesenchymal stem cells and lineage-uncommitted pluripotent mesenchymal stem cells. The present study was undertaken to assess the distribution of these stem cells in the connective tissues throughout various regions of the body. Day 11 chick embryos were divided into 26 separate regions. Heart, limb skeletal muscle, and limb dermis were included as control tissues. Cells were harvested enzymatically and grown using conditions optimal for the isolation, cryopreservation, and propagation of avian mesenchymal stem cells. Cell aliquots were plated, incubated with various concentrations of dexamethasone, and examined for differentiated phenotypes. Four recurring phenotypes appeared in dexamethasone-treated stem cells: skeletal muscle myotubes, fat cells, cartilage nodules, and bone nodules. These results suggest that progenitor mesenchymal stem cells and putative pluripotent mesenchymal stem cells with the potential to form at least four tissues of mesodermal origin have a widespread distribution throughout the body, being located within the connective tissue compartments of many organs and organ systems.