Herpes specific and alpha DNA polymerase in nuclear envelope of BHK infected cells

Human serum contains an ultrafiltrable factor (350 < M.V. < 700) which stimulates sulphation activities of native, or purified somatomedin A of either small or high molecular weight. The factor is heat stable, resists protease hydrolysis but is destroyed by strong acidic hydrolysis. It is not extractible by chloroform. It restores somatomedin activities of conserved fractions and allows good conditions of bioassays of purified fractions. This factor is not a known amino-acid, a polyamine, vitamin A, zinc, T4 or T3. It stimulates somatomedin activity equally if added together with the somatomedin, or if added before (and removed) the adding of somatomedin.     

Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: Relation to human somatomedins and insulin

The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component ot adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.     

Ontogeny of somatomedin during development in the mouse. Serum concentrations, molecular forms, binding proteins, and tissue receptors

The purpose of this study was to assess the ontogeny of serum concentrations and molecular forms of somatomedin during fetal and postnatal development and to define the changes in serum binding proteins for somatomedin-C during various stages of development. The finding that fetal, placental, and decidual mouse tissues possess receptors for somatomedin suggests a role for somatomedin in fetal growth and possibly in the maintenance of pregnancy. Serum somatomedin-C was measured using a highly specific, heterologous radioimmunoassay (RIA) and a less specific membrane binding assay (MBA) which is more sensitive to the influence of somatomedins other than somatomedin-C. The assays were validated for mouse serum by showing that serum concentrations were reduced in genetically growth hormone-deficient mice and in hypophysectomized mice and were increased by growth hormone therapy. As in the human, the RIA measures only a portion of the somatomedin-C present in mouse serum. This “covering up” of somatomedin is attributed to the presence of serum binding proteins and is corrected by treatment of serum samples with acid. By both RIA and MBA, serum somatomedin concentrations are low in fetal and newborn mice, begin to rise in the fourth postnatal week, and reach adult values by 7 weeks of age. The chromatographic pattern of adult mouse serum on Sephacryl 200 is similar to that observed with human sera: The immunoreactive material elutes at apparent molecular weights of 140,000 and 30,000–40,000. The elution profile of ¹²⁵I-labeled somatomedin-C bound to components of serum is nearly identical to the pattern of endogenous activity. As with human serum, somatomedin-C in acidified mouse serum elutes at a lower molecular weight, coincident with insulin and purified somatomedin-C. Maternal serum somatomedin declines in the last half of gestation at the time when placental lactogen levels rise. Along with the absolute decline in somatomedin content is the appearance of unsaturated sites on somatomedin binding proteins. These findings are unexpected and unexplained since somatomedin rises late in pregnancy in humans and several lines of evidence suggest that placental lactogen has the capacity to stimulate somatomedin production. We previously have presented evidence that explants of multiple fetal mouse tissues synthesize somatomedin-C. The present study shows that the immunoreactive somatomedin-C in fetal mouse serum shares identical characteristics with those reported previously for media obtained from mouse liver explants. It seems possible that somatomedin's actions are exerted primarily at or near its site of production and that circulatory levels do not reflect the importance of somatomedin-C on fetal growth. While elucidation of the dramatic developmental changes in serum content and molecular forms of somatomedin-C and in somatomedin binding proteins may be essential to clarifying the role of somatomedin on fetal growth, proof that somatomedin stimulates fetal growth will depend in large part on studies of its biological actions on fetal tissues.     

Decrease in serum receptor-reactive somatomedin in diabetes.

Somatomedin in rat serum has been measured by a sensitive radioreceptor assay using 125I-labelled human somatomedin and human placental membrane. In rats made diabetic with strepotzotocin, receptor-reactive somatomedin levels were decrease by up to 75%. The decrease followed the time course of increasing serum glucose and occurred to the same extent in rats aged between 4 and 40 weeks. Endogenous serum receptor-reactive somatomedin appeared exclusively in high molecular weight fractions on gel chromatography. In diabetes the decreased somatomedin was due to a fall in this high molecular weight activity, but was not accompanied by a fall in somatomedin binding protein. These results suggest a role for insulin in maintaining serum somatomedin levels.     

The influence of early nutrition on growth and the circulating levels of immunoreactive somatomedin A.

The effect of nutrition during early development on growth and serum immunoreactive somatomedin A was examined in rats by rearing in small or large liters. At weaning, body weight, brain weight, liver weight and DNA content was significantly reduced in rats whose nutrition was restricted by rearing in large litters. Brain DNA content was significantly altered. Serum somatomedin A was significantly reduced in the growth-retarded rats as compared to those whose growth was enhanced by rearing in small litters. Similarly, maternal serum somatomedin A was significantly reduced in rats nursing large litters.     

Pygmy mouse fibroblasts in tissue culture: evaluation of multiplication stimulating activity (MSA) receptors and growth responses to MSA

The pygmy mouse has been proposed as a model for growth hormone resistance; it has normal serum somatomedin levels and does not respond to growth hormone treatment. In order to determine if the growth impairment is caused by a defect in somatomedin binding or in postreceptor action of somatomedin we compared fibroblasts derived from pygmy mice with those from normal appearing littermates. Using multiplication-stimulating activity (MSA) as a model somatomedin we found a normal Ka of binding to the cell surface MSA receptor but a significantly increased number of MSA receptors on the fibroblasts derived from pygmy mice. Studies of thymidine incorporation into DNA failed to demonstrate a difference between pygmy and normal fibroblasts in their responses to MSA alone, but there was a significantly greater thymidine incorporation into the DNA of normal fibroblasts when both competence factor (platelet-derived growth factor) and progression factors (somatomedins and growth hormone deficient platelet-poor plasma) were present in the test medium. On the other hand, cell proliferation studies did not demonstrate a consistent difference in the growth rate of normal versus pygmy fibroblasts. The data support the conclusion that the imparied growth of the pygmy mouse in vivo may be caused by factors which lie outside of the growth hormone-somatomedin pathway.     

Direct mitogenic effects of human somatomedin on human embryonic lung fibroblasts

The ability of purified basic somatomedin to reinitiate cell division in nondividing cultures of human embryonic lung fibroblasts (WI-38) maintained in serum-free medium was determined in order to assess the direct mitogenic effect of this substance on mammalian tissue. Resting cultures were prepared by incubation of the cells in serum-free medium for 48–72 hours. Addition of partially purified somatomedin resulted in cellular hypertrophy, DNA synthesis and cell division with a time course similar to that seen when serum was added. Although cells divided in response to physiological concentrations of somatomedin, doses up to 100x in excess of this did not produce as much cell division as 10% fetal calf serum. Addition of fresh medium containing somatomedin to cells previously stimulated by somatomedin failed to induce further cell division. A highly purified somatomedin preparation also stimulated cell division.     

Determination of the major zinc fractions in human serum by ultrafiltration

In this paper we report a method for measuring ultrafiltrable zinc in human serum by electrothermal atomic absorption spectrophtometry. We show also that ultrafiltration permits to determine alpha-2 macroglobulin bound zinc and losely bound zinc if a strong zinc ligand (EDTA) is added to serum before ultrafiltration. This last fraction, after deduction of ultrafiltrable zinc, represents roughly all albumin bound zinc. In 20 controls we found that ultrafiltrable zinc amounted 0.311 μmol/L (S.D.=0.117 μmol/L), alpha-2 macroglobulin bound zinc 3.08 μmol/L (S.D.=0.221 μmol/L), and albumin bound zinc 12.11 μmol/L (S.D.=1.95 μmol/L). Our method needs only a small volume of serum, it is simple and rapid but also very accurate and reliable. The losely bound fraction is very dynamic and, representing the physiologically active part of serum zinc, it could be a good marker of zinc deficiency.     

Binding protein of somatomedin A in serum from men and rats

After gel chromatography of human and rat serum at pH 7.4, all endogenous somatomedin A was recovered in the high molecular weight range. The largest peak was found in the gamma-globulin (II) region and the next largest peak found in the albumin region (III). The amounts of somatomedin A in the peak II region increased in serum from acromegalies and decreased in serum from growth hormone deficient patients. Four radioactive peaks were observed after gel chromatography of serum incubated with 125I-somatomedin A. Only the two peaks corresponding to peaks II and III out of the four peaks were displaced by adding 50 microgram of partially purified cold somatomedin A. The radioactivity of peak II decreased in sera from growth hormone deficient patients and increased after growth hormone administration. These observations support the hypothesis that the growth hormone regulates not only somatomedin A but also its carrier protein.     

Immunological cross-reactivity of multiplication-stimulating activity polypeptides

The immunoreactivity of the multiple species of multiplication-stimulating activity (MSA) purified from medium conditioned by a rat liver cell line (BRL-3A) has been examined. Antibodies were raised in rabbits following immunization with MSA II polypeptides. Subpopulations of antibodies were purified from one antiserum using DEAE-cellulose chromatography. One antibody subpopulation recognized common antigenic determinants on MSA I and MSA II polypeptides; whereas a second antibody subpopulation recognized common determinants on MSA I, II, and III polypeptides. In a radioimmunoassay utilizing 125I-MSA III-2 and a purified antibody subpopulation, the human somatomedins (somatomedin A, insulin-like growth factor I and II) showed weak, but significant cross-reactivity: insulin-like growth factor II was 10% as potent as MSA II. By contrast, somatomedin partially purified from rat serum, insulin, growth hormone, epidermal growth factor, nerve factor, and fibroblast growth factor, showed no reactivity in the radioimmunoassay.     

Insulin-like growth factors, IGF-1, IGF-2 and somatomedin C trigger cell proliferation in mammalian epithelial cells cultured in a serum-free medium

A single insulin-like growth factor which constitutes part of a defined serum-free medium is sufficient to stimulate DNA synthesis and mitosis in mammalian lens epithelial cells. Rabbit lenses were cultured in KEI-4, a medium which mimics rabbit aqueous humor, or in KEI-4 containing insulin growth factor I (IGF I), insulin growth factor II (IGF II) or somatomedin C. The magnitude of DNA synthesis and mitosis was evaluated on whole mount preparations of the epithelium at various times of culture. IGF I and II, the most highly purified of the insulin-like growth factors, and somatomedin C were equipotent lens mitogens, were active at the ng level, were more mitogenic toward lens epithelial cells than insulin, and initiated cell proliferation throughout the normally amitotic central region of the lens epithelium. The time course of the mitotic response elicited by the insulin-like growth factors was identical to that noted in lenses cultured in medium supplemented with serum or insulin. The present results, coupled with those of other investigators, suggest that insulin-like factors may regulate cell division in the mammalian lens in vivo.     

Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells

The activity in platelet-poor plasma that allowed density-arrested BALB/c-3T3 cells rendered competent by a transient exposure to platelet-derived growth factor (PDGF) to traverse G1 and enter the S phase has been termed progression activity. Epidermal growth factor (EGF) and somatomedin C-supplemented medium was shown to be capable of replacing the progression activity of 5% platelet-poor plasma (PPP) for competent density-inhibited BALB/c-3T3 cells. Exposure of competent cells to medium supplemented with EGF and somatomedin C reduced the 12 h minimum G1 lag time found in plasma-supplemented medium by 2 h. It is suggested that the reduction in the minimum time required for progression through G1 is due to the availability of free, unbound somatomedin C. Complete G1 traverse required both EGF and somatomedin C; however, the traverse of the last 6 h of G1 and entry into the S phase required only somatomedin C. Though EGF and somatomedin C could replace the G1 phase progression activity of plasma, medium supplemented with EGF and somatomedin C did not support complete cell cycle traverse or growth of sparse cultures of BALB/c-3T3 cells.     

Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion.

cDNA clones coding for human S-protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S-protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S-protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of S-protein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino-terminal residues 42 are identical with the so-called somatomedin B peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S-protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways.     

Production and isolation of recombinant somatomedin C

High-level production of a growth promoting peptide hormone somatomedin C (insulin-like growth factor I) has been achieved using recombinant DNA techniques in Escherichia coli. We found a new structural protein, designated as LH, to stabilize somatomedin C in vivo, and constructed expression vectors for somatomedin C fusing to LH through a methionine and through a tryptophan, respectively. Each of the fused proteins was produced at approximately 4.5 X 10(5) molecules per single E. coli cell and comprised more than 20% of the total cellular proteins. Somatomedin C was obtained in high yield by limited cyanogen bromide degradation of the methionine-type fused protein, in spite of somatomedin C itself having a Met at the 59th position, followed by renaturation of the resultant reduced peptide. The tryptophan-type fused protein was also converted to the peptide hormone by treating with 3-bromo-2-nitrophenylsulphenyl skatole or N-chlorosuccinimide. The recombinant somatomedin C obtained by these procedures was identical with the native one in amino acid sequence as well as in biological activity of stimulation of DNA synthesis in BALB/c 3T3 cells.     

The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in ovarian cells

The actions of insulin and somatomedin C (insulin-like growth factor I) on cholesterol side-chain cleavage activity and the synthesis of cytochrome P-450scc and adrenodoxin were investigated in primary cultures of swine ovarian (granulosa) cells. Nanomolar concentrations of pure human somatomedin C stimulated biosynthesis of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Moreover, in the presence of exogenous sterol substrate for cholesterol side-chain cleavage, somatomedin C significantly enhanced pregnenolone biosynthesis in a time- and dose-dependent manner. This augmentation of functional cholesterol side-chain cleavage activity was accompanied by a dose-dependent (2-16-fold) increase in [35S]methionine incorporation into specific immunoprecipitable cytochrome P-450scc and adrenodoxin. Micromolar concentrations of insulin (but not proinsulin or desoctapeptide) also induced synthesis of cholesterol side-chain cleavage constituents by 4-7-fold. These results demonstrate that an insulin-like growth factor, somatomedin C, exerts discrete differentiating effects on ovarian cells characterized by increased synthesis of immunospecific cytochrome P-450scc and adrenodoxin. Thus, we infer that somatomedin C may serve a critical role in the differentiation of steroidogenic cells in the mammalian ovary.     

A role for somatomedin C as a differentiating hormone and amplifier of hormone action on ovarian cells: studies with synthetically pure human somatomedin C and swine granulosa cells

Isolated swine granulosa cells incubated in chemically defined medium in vitro responded to synthetically pure human somatomedin C/IGF-I in a dose and time-dependent fashion with increased pregnenolone, progesterone and estradiol biosynthesis. These stimulatory actions were not mimicked by growth hormone, proinsulin, desoctapeptide insulin, epidermal growth factor, or fibroblast growth factor. Moreover, somatomedin C/IGF-I augmented the steroidogenic response of granulosa cells to exogenously supplied sterol substrate in the form of low-density lipoprotein, and amplified the stimulatory actions of the classical ovarian effector hormones, estradiol and follicle-stimulating hormone, in a synergistic fashion. The ability of somatomedin C/IGF-I to stimulate estradiol production on the one hand, and to act synergistically with estradiol to stimulate progesterone biosynthesis on the other hand, suggests a unique intrafollicular mechanism for amplifying progestin biosynthetic capacity in granulosa cells.     

Purification of a high molecular weight somatomedin binding protein from human plasma

The somatomedins insulin-like growth factor I and II (1,2) are in serum bound to high-molecular weight binding proteins (6,7,8). By use of a four step chromatographic procedure a somatomedin binding protein was isolated from outdated human plasma. Exclusion chromatography on Sephadex G-200 disclosed a molecular weight of 150 kDa. After lyophilization however, the binding activity was found in a lower molecular weight range of 35-45 kDa. A partial amino acid sequence analysis of the lyophilized material revealed a possible N-terminal sequence of Ala-Pro-Trp. This sequence is identical to the N-terminal sequence of the 35 kDa somatomedin binding protein previously isolated from human amniotic fluid (16).     

Plasma GH and somatomedin C responses to single and repeated administrations of human growth hormone releasing factor (hGRF)

Plasma growth hormone (GH) and somatomedin C responses to single and repeated administrations of synthetic human growth hormone releasing factor (hGRF-44) were studied in 29 patients with GH deficiency. hGRF-44 administered by single iv bolus (10 micrograms/kg), repeated iv boluses (50 or 100 micrograms, once a day), repeated iv infusions (2.5 micrograms/min for 90 min, once a day), and/or repeated im injections (100 micrograms, twice a day) for three to six consecutive days. Ten of 16 patients had plasma GH responses to a single iv bolus injection, i.e., their plasma GH increased above 5 ng/ml or twice the basal level. However none of them showed a plasma somatomedin C increase. To repeated iv bolus injections and repeated iv infusions of hGRF-44, 7 of 17 patients showed plasma GH responses, however in none of 7 patients did somatomedin C increase. Plasma somatomedin C did not increase after repeated im administrations of hGRF-44 for 5 days. Plasma somatomedin C increase was observed in two patients, from 0.32 to 0.54 U/ml and from 0.16 to 0.46 U/ml, in response to repeated iv boluses and repeated iv infusions, respectively. These results suggest that hGRF-44 stimulates plasma GH secretion in some patients with GH deficiency, however the increases are not enough to stimulate somatomedin generation.     

A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts

A polypeptide fraction with multiplication-stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum-free medium conditioned by the growth of a line of rat liver cells. The specific multiplication-stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication-stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non-suppressible insulin-like activities, but did not have anti-trypsin activity. The rat liver cell multiplication-stimulating activity resembled both multiplication-stimulating activity from calf serum and somatomedin.     

Effect of the variations of female sex hormones during the menstrual cycle upon serum somatomedin and growth-promoting activity

Serum growth-promoting activity measured upon lymphocytes, sulfation activity and radioimmunoassayable somatomedin C (Sm-C) levels were measured in sera from women during the menstrual cycle. The data showed that: estradiol, progesterone, LH or FSH added in vitro do not increase the 3H-thymidine uptake into lymphocytes; the serum thymidine activity decreases during the luteal stage of the cycle, and is negatively correlated with the progesterone levels; the sulfation factor and Sm-C levels do not have significant variations during the menstrual cycle, and the GH maximum values are attained during the luteal stage.