Stimulation Of Amino Acid Incorporation Research Articles

The physiology of growth hormone (GH) in adults: translational journey to GH replacement therapy.

The fact that growth hormone (GH) plays an important role in health after the cessation of growth requiring replacement therapy in adult life has only been recognised in the last three decades. This has only been made possible by recombinant technology providing GH supplies required to undertake investigations in the physiology of GH action and the benefits of replacement therapy in patients identified by rigorously validated diagnostic tests for GH deficiency (GHD). Human studies have revealed important regulatory roles in substrate metabolism, sodium homeostasis, body composition, and physical function. GH-induced anabolism is achieved by stimulating amino acid incorporation into protein while reducing oxidative loss simultaneously enhancing lipid utilisation by stimulating fatty acid oxidation and reducing lipid storage. Sodium and fluid retention are enhanced by activating the renin-angiotensin system and distal renal tubular reabsorption. GH stimulates the aerobic and anaerobic energy systems that underpin muscle and cardiovascular function. These pleiotropic actions explain the clinical picture of increased adiposity, reduced lean mass, and impaired physical and psychological function in the GHD adult, all of which are reversed when GH is replaced. Women require a greater replacement dose of GH than men. This is because androgens enhance while oestrogens attenuate GH action. The oestrogen effect is route-dependent, occurring with oral delivery blunting the liver-mediated actions of GH by directly inhibiting GH receptor signalling, global experience spanning over 30 years has attested to the safety, efficacy, and benefits of replacement therapy for adults with GHD.

Surfactant stimulation of growth in the nematode Caenorhabditis elegans

Size fractionation has been used to isolate L1 L2 larvae from mixed cultures of the nematode, Caenorhabditis elegans. Worm lengths have been compared during growth in synchronized liquid and agar cultures. Supplementation of liquid S medium with 10 ppm surfactant (Pluronics F-68, F-127, F-38, L-35; Tween-20 or Triton X-100) promoted a significant stimulation of growth over three days in all cases. Because of possible poor nutrition and/or aeration in liquid culture, experiments were repeated using the same surfactants in standard NGM agar. Four surfactants again stimulated worm growth significantly whereas two (Tween-20 and Pluronic F-68) did not. Differences between these surfactants were also demonstrated with respect to: (i) toxicity; (ii) induction of stress responses in a transgenic hsp/reporter strain; and (iii) stimulation of amino acid incorporation into soluble protein both initially and after 41 h of surfactant treatment. These surfactants, and in particular, Pluronic F-127, are potentially valuable as culture supplements for enhancing nematode larval growth. Possible mechanisms for growth promotion by surfactants are discussed in light of the fixed somatic cell lineage and the fact that Pluronic F-127 did not speed up maturation from L4 larvae into egg-bearing adults.

Effects of iodide on thyroglobulin biosynthesis in FRTL-5 cells.

Iodide inhibits several thyroid parameters through an organic intermediate, and this process has been related to thyroid autoregulation. The aim of this study was to determine the effect of iodine on thyroglobulin (Tg) synthesis in the rat thyroid cell line FRTL-5. TSH stimulated amino acid incorporation into the cells by 400% and iodine had no effect on this parameter. No effect of TSH or iodide on [35S] methionine incorporation into protein was found under our experimental conditions (approximately 80% of total [35S]methionine incorporated was found in TCA-precipitable material). TSH caused an increase in Tg synthesis, after 1 h, while iodide partially blocked the effect of TSH (control 6.4% of TCA precipitable radioactivity; TSH 10.7%; iodide 8.4%). After 24 h, the protein released into the medium was measured. TSH stimulated total protein liberation and iodide inhibited this parameter. TSH stimulated total RNA content, and iodide caused an inhibition. Northern analysis did not show inhibition by iodide of TSH-stimulated Tg mRNA levels. The present results show an inhibitory effect of excess iodide on TSH-stimulated thyroglobulin biosynthesis in FRTL-5 cells.

Insulin-stimulated protein synthesis in submandibular acinar cells: interactions with adrenergic and cholinergic agonists.

The effects of insulin and secretory agonists on amino acid incorporation into submandibular gland proteins were studied using isolated acinar cell aggregates. Insulin stimulated the incorporation of 3H-leucine into TCA-precipitable proteins in a rapid, dose-dependent manner (half-maximal response at 1 nM). Isoproterenol, a beta-adrenergic agonist, also stimulated amino acid incorporation, and this effect was mimicked by both dibutyryl cAMP and IBMX, a phosphodiesterase inhibitor. Although insulin further stimulated incorporation in the presence of isoproterenol and IBMX, no additional increase in the rate of synthesis was observed after stimulation by dibutyryl cAMP. High concentrations of carbamylcholine, a cholinergic agonist, inhibited both basal and insulin-stimulated incorporation. At low concentrations, however, carbamylcholine stimulated synthesis, and the effects of insulin and carbamylcholine were additive. A23187, a calcium ionophore, also inhibited 3H-leucine incorporation and insulin stimulation, but in contrast to carbamylcholine, low concentrations of A23187 neither inhibited nor enhanced the rate of synthesis. Thus, protein synthesis in the rat submandibular gland is regulated by both insulin and neurotransmitters. Whereas beta-adrenergic stimulation appears to be mediated through cAMP, the intracellular signals mediating the actions of insulin and cholinergic agonists remain to be elucidated.

Molecular basis for heterosis for myelin basic protein content in mice.

Poly(A)+ mRNA was isolated from the brains of C57BL/6J (B6), DBA/2J (D2), and F1 hybrid mice (B6 X D2) of 16-17 days of age. The yield of polysomal RNA, both poly(A)+ and poly(A)-, from the three strains of mice was comparable. When translated in vitro in a reticulocyte lysate system, the mRNA preparations had the same efficiency with respect to stimulation of amino acid incorporation into protein. However, a significant heterotic effect was seen for the production of myelin basic protein (MBP) by the mRNA from the F1 mice. That is, the fraction of protein synthesized as MBP was greater for the F1 hybrid than for either parental strain. The distribution of the form of MBPs was not different among the three strains. We therefore believe that heterosis for brain MBP content in the F1 hybrid may be regulated at the transcriptional or post-transcriptional level.

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4–5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3–3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [γ- 32P]ATP results in the phosphorylation of a protein of M r 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2α. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.

Interaction of spermidine with viral RNA and its influence on protein synthesis

Addition of spermidine to a cell-free protein synthesizing system from wheat germ programmed with total brome mosaic virus (BMV) RNA resulted in a several-fold stimulation of amino acid incorporation. Increasing the spermidine concentration in the system led to inhibition of the overall protein synthesis, but the production of longer polypeptides was inhibited much more than that of the coat protein (shorter product). Analysis of the products synthesized under direction of BMV RNA 3 (longer product) and RNA 4 (coat protein) revealed that optimal translation of RNA 3 occurred at a much lower concentration of spermidine than that of RNA 4. Binding experiments with radioactive spermidine and BMV RNAs showed that the saturation of spermidine binding is achieved at a lower concentration of spermidine for RNA 3 than for RNA 4, which may suggest that the structure of RNA 4 is more compact than that of RNA 3. Taking into account the binding obtained at a spermidine concentration corresponding to optimal conditions of protein synthesis, it may be concluded that the optimum translation of these two mRNAs occurs when there is a similar level of RNA charge neutralisation, which implies a similar level of RNA structure stabilisation.

Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action.

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

Ca2+ and hormones interact synergistically to stimulate rapidly both prolactin production and overall protein synthesis in pituitary tumor cells.

Effects of Ca2+ and hormones on short-term protein synthesis were examined utilizing intact Ca2+-depleted and Ca2+-restored GH3 pituitary tumor cells as a model system. Amino acid incorporation by cells in complete growth medium during short incubations was markedly reduced by EGTA concentrations in excess of Ca2+. Thyrotropin-releasing hormone (TRH) rapidly enhanced amino acid incorporation and prolactin production, with both effects being reserved by EGTA in excess of extracellular Ca2+ or prevented by cellular Ca2+ depletion. Epidermal growth factor and phorbol myristate acetate (PMA) also stimulated amino acid incorporation and prolactin production; absolute increases in protein synthesis provided by these agents were significantly greater in Ca2+-restored than in Ca2+-depleted preparations. TRH and PMA concentrations which raised prolactin production were identical to those increasing the rate of amino acid incorporation into overall protein. The extracellular Ca2+ concentration dependencies of amino acid incorporation and prolactin production were similar and were unchanged by hormone. PMA, the most efficacious of the agents tested, and Ca2+ promoted incorporation of amino acid into the same spectrum of proteins. Stimulation of protein synthesis by hormones was not attributable to alterations in amino acid uptake, attachment to substrata, hormone binding, protein catabolism or transcription. Trifluoperazine selectively prevented the stimulation by Ca2+ of amino acid incorporation and prolactin production. Unlike total prolactin, the total protein content of GH3 cells during these short incubations was not altered by Ca2+, hormones or trifluoperazine. It is proposed that hormones and Ca2+, which have been demonstrated to regulate prolactin secretion and prolactin mRNA transcription in GH3 cells, also exert translational controls which serve to facilitate the overall expression of the prolactin gene.

Effect of exogenous addition of hemin on the biogenesis of mitochondrial membranes during glucose repression in Saccharomyces cerevisiae

Exogenous addition of hemin alleviated glucose repression by promoting mitochondrial membrane functions. It prevented the release of mitochondrial proteins into the cytosol by decreasing the activities of phospholipase C and phospholipase D. It restored oligomycin sensitivity of mitochondrial ATPase associated with repressed mitochondria. It enhanced cardiolipin content twofold, and decreased the sterol: phospholipid ratio. Studies on the amino acid incorporation into isolated mitochondria showed that hemin can also promote biogenesis of the organelle by stimulating amino acid incorporation into mitochondrial proteins, and this stimulation appeared to be mediated through some cytosolic factor(s).

Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells.

The incorporation of methionine, lysine, and leucine into protein was studied in Ca2+-depleted and Ca2+-restored preparations of C-6 glial tumor cells in minimal medium. Although incorporation proceeded at linear rates in both preparations for more than 1 h and into the same spectrum of proteins, Ca2+-restored cells incorporated amino acid 5- to 10-fold more rapidly than Ca2+-depleted cells. Addition of approximately 200 microM Ca2+ in excess of chelator was required to achieve maximal rates of incorporation in Ca2+-depleted preparations. Stimulation by Ca2+ was rapid in onset (several minutes) and slowly reversible by chelator. Ca2+ was uniquely potent and specific among physiologically occurring cations in conferring such stimulation. Stimulation of amino acid incorporation by Ca2+ occurred over a broad range of pH and osmolarities and was facilitated by Mg2+. The effects of Ca2+ in stimulating amino acid incorporation were not traceable to changes in cAMP metabolism, amino acid uptake, protein catabolism, cell ATP or GTP content, or aminoacylation of transfer RNA. Actinomycin D (1 microgram/ml) did not block the stimulatory effects of Ca2+ although puromycin and cycloheximide did. The stimulatory effects of Ca2+ on protein synthesis were not restricted to C-6 in minimal medium. Protein synthesis was reduced by ethylene glycol bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid 40 to 75% in C-6 glioma, GH3 pituitary tumor, PC-12 adrenal tumor, N2A neuroblastoma, and HeLa cells incubated under simulated growth conditions with various enriched media and sera. Ca2+-depleted S49 lymphoma, CHO ovarian tumor, and normal, dispersed chicken embryo cells in enriched medium responded to Ca2+ restoration with increased rates of protein synthesis as did collagenase-dispersed normal rat liver cells in minimal medium. Protein synthesis in rabbit reticulocyte lysates was also inhibited by Ca2+-selective chelators or by Ca2+ removal by parvalbumin affinity chromatography and the inhibition was reversed by Ca2+. These findings are consistent with the existence of a Ca2+ requirement in the translational phase of protein synthesis in eukaryotic cells.

Anabolic principles of Aconitum roots

The methanol extracts of raw and processed roots of Aconitum carmichaeli were shown to stimulate amino acid incorporation into mouse liver protein after approx. 10 h of ingestion. The extract of the raw roots was fractionated by monitoring the anabolic activity to furnish the aconitine alkaloids as active principles, among which mesaconitine exhibited the strongest activity. Amino acid incorporation into liver protein in the mesaconitine treated mice was inhibited by actinomycin D to the level of normal mice. Long-term administration of mesaconitine induced no reinforcement of the anabolic activity in mouse liver. Examination of the anabolic activity in liver, spleen, kidney, testis and serum revealed that mesaconitine potentiated protein synthesis only in liver and rather reduced it in kidney.

Experimental methyl mercury neurotoxicity: Similar in vivo and in vitro perturbation of brain cell-free protein synthesis

Perturbation of brain protein synthesis by methyl mercury chloride (MeHg) was compared in vivo and in vitro. MeHg-stimulated and/or inhibited brain cell-free protein synthesis following in vivo or in vitro administration. Although pretreatment with GSH protected the postmitochondrial supernatant (PMS) from the in vitro inhibition, direct addition of -SH compounds did not reverse the in vivo or in vitro perturbations in synthesis induced by MeHg. Inhibition of synthesis induced by both in vivo and in vitro methyl mercury administration resulted in inactivation of component(s) in brain pH 5 enzymes. Stimulation of amino acid incorporation following in vivo administration of MeHg was apparently associated with the ribosome fraction, but in vitro preincubation of PMS with MeHg produced stimulation associated with the pH 5 enzyme fraction. A model of MeHg neurotoxicity was proposed providing a common molecular locus of interaction in vivo and in vitro.

The Effects of Electric Currents on ATP Generation, Protein Synthesis, and Membrane Transport in Rat Skin

Direct electric currents ranging from 10 microA to 1000 microA increase ATP concentrations in the tissue and stimulate amino acid incorporation into the proteins of rat skin. The amino acid transport through the cell membrane, followed by the alpha-aminoisobutyric acid uptake, is stimulated between 100 microA and 750 microA. The stimulatory effects on ATP production and on amino acid transport, apparently mediated by different mechanisms, contribute to the final increased protein synthesizing activity. DNA metabolism followed by thymidine incorporation remains unaffected during the course of current application. The effects on AtP production can be explained by proton movements on the basis of the chemiosmotic theory of Mitchell, while the transport functions are controlled by modification in the electrical gradients across the membranes.

Effect of cytosol on mitochondrial protein synthesis in vitro during cardiac hypertrophy

Studies on amino acid incorporation into isolated mitochondria in vitro were carried out in normal and hypertrophic hearts obtained after aortic constriction. The incorporation of labeled amino acids in vitro was triggered immediately after 6 h of operation as an early response to the induced work load and at further time periods the incorporation increased rather slowly and reached a maximum at 8 days after aortic constriction. At a further time period when the hypertrophy developed to an advanced stage, the incorporation was drastically reduced. The cytosol obtained from normal and hypertrophic hearts had a stimulatory effect on the labeled amino acid incorporation in vitro into isolated mitochondria from normal hearts. This stimulatory effect was maximum with cytosol obtained from 40% hypertrophic animals. Partial characterization of the factors involved in the stimulation of amino acid incorporation in vitro revealed the involvement of heat-labile, nondialyzable, and ammonium sulfate-precipitable protein stimulatory factors.

Calcitonin stimulates amino acid incorporation into plasminogen activator by cultured renal tubular cells

Received 30 July 1981 1. Introduction A cultured epithelial cell line (LLC-PK1) derived from porcine kidney has receptors for calcitonins and responds to these hormones with an increase in the cellular content of CAMP [ 1,2] and activation of a CAMP-dependent protein kinase [3]. The LLC-PK1 cells had been selected and maintained in (41 because of their ability to produce large amounts of plasmin- ogen activator. The synthesis and secretion of this enzyme in several other cell systems is modulated by specific hormones, some of which act by increasing cellular levels of CAMP [5,6]. Calcitonin also markedly stimulates plasminogen activator production by LLC- PK1 cells and decreases their replication [7]. Here, we show that calcitonin increases amino acid incorpora- tion into proteins secreted by LLC-PK1 cells. The increased incorporation is mainly into a protein which on polyacrylamide gel electrophoresis has an estimated M, of m 45 000 and has plasminogen activator activity. 2. Materials and methods 2.1.

Differential response to growth factor by rat mammary epithelium plated on different collagen substrata in serum-free medium.

Primary cultures of rat mammary epithelial cells proliferate and synthesize basement membrane collagen (type IV collagen) in a serum-free medium supplemented with epidermal growth factor (EGF), hydrocortisone or dexamethasone, insulin, transferrin, and Pedersen fetuin. The growth response of the cells to EGF and glucocorticoids but not to insulin or transferrin varies depending on the substratum on which the cells are plated. Cell growth is 4 times more sensitive to omission of EGF or glucocorticoid on type I collagen or plastic substratum than on type IV collagen substratum. The mechanism by which these two growth factors differentially affect cell growth appears to be linked to an increase in type IV collagen synthesis and a stabilization of secreted type IV collagen in the extracellular matrix. Glucocorticoids suppress the elaboration of type IV collagenolytic activity by the cells whereas EGF stimulates amino acid incorporation into type IV collagen. The results suggest that EGF and glucocorticoids affect mammary epithelial cell growth by facilitating the accumulation of the appropriate cell substratum.

The characteristics of the cell-free translation of mRNA from cells infected with the herpes virus pseudorabies virus.

The translation in vitro of mRNA from pseudorabies virus infected cells was studied using systems derived from wheat germ and from rabbit reticulocyte. The mRNA was shown by molecular hybridization to contain sequences complementary to virus DNA. Products of in vitro translation co-migrating with virus proteins on polyacrylamide gel electropherograms were detected and the major immune precipitation. Optimum conditions for the stimulation of amino acid incorporation in vitro were determined and found to be similar for mRNA from both infected and mock-infected cells.

Stimulation of encephalomyocarditis virus RNA translation in wheat embryo extracts by rabbit reticulocyte ribosomal wash.

Encephalomyocarditis virus RNA induced a low level of amino acid incorporation in wheat embryo extracts, but the incorporation did not result in the formation of any viral proteins. However, if wheat embryo extracts were supplemented with a ribosomal wash fraction from rabbit reticulocytes, a large stimulation of amino acid incorporation and the formation of encephalomyocarditis virus-specific proteins were obtained. Among the proteins synthesized were capsid precursor proteins which can be processed by an encephalomyocarditis virus-coded protease to produce the viral capsid proteins.

Isolation of ganglion cells from the retina

A microdissection technique was developed to isolate cellular layers of the goldfish retina. Three regions of the neural retina, the photoreceptor-interneuron layer (PL), the vascular layer (VL), and the ganglion cell layer (GCL) were recovered. A cleavage plane between the PL and GCL occurred at the level of the inner plexiform region as shown by light microscopy. Scanning electron microscopy found the predominant cells of the PL to include rods and cones while the VL contained a capillary network that arose from the retinal artery. The GCL consisted of pear-shaped ganglion cells about 5 μm in size with single, unbranched axons with diameters of about 0.5 μm. This preparation was virtually free of other cell types or debris. Transmission electron microscopy demonstrated that axons of the GCL were unmyelinated in contrast to those fibers distal to the optic nerve head. On the basis of morphology, microdissection could isolate a single population of retinal neurons free of glia. Biochemical indicators were used to determine the degree of separation of the GCL from the PL and VL. Markers of the cholinergic system, acetylcholinesterase and α-bungarotoxin binding, predominate only within the PL. These findings are consistent with known histochemical features of the retina and suggest that the GCL is not contaminated by the neighboring photoreceptor-interneuron region. Ganglion cell axons distal to the optic nerve head are myelinated and contain high levels of the marker enzyme 2′,3′-cyclic nucleotide-3′-phosphodiesterase. In contrast, this enzymatic activity is not detected within the unmyelinated axons of the glia free GCL preparation. To examine retinal events during axonal regeneration, transection of ganglion cell axons was carried out at the level of the optic tract. Axotomy led to stimulated amino acid incorporation detected within the GCL and not other regions of the retina. This specific response of the GCL was observed both in vivo and in vitro indicating that isolated ganglion cells remain intact and biologically active. Microdissection provides, therefore, a population of retinal ganglion cells that may be used to analyze the biochemical events of neuronal regeneration.