Synthesis Of Other Proteins Research Articles

Inhibition of collagen synthesis in cultured cells by hypertonic culture medium

The synthesis of collagen under conditions in which polypeptide chain initiation is selectively inhibited by medium hypertonicity was compared to the synthesis of other proteins in chick embryo leg bone cells in monolayer cultures. Three different approaches showed that collagen synthesis is far more sensitive than the majority of other cellular proteins to the hypertonic initiation block. In marked contrast, the synthesis of an unidentified protein, migrating with an apparent molecular of 45,000 to 50,000 is particularly resistant to hypertonicity. The effects of hypertonic conditions were found to be readily reversible upon restoration of isotonicity. Since these suboptimal growth conditions can decrease the amount of collagen synthesized relative to total protein synthesis, they provide an experimental model for the study of the translational control of the synthesis of collagen and other proteins.

The effect of zinc deficiency on the metabolism of retinol-binding protein in the rat.

Abstract Zinc-deficient rats have very low plasma vitamin A levels in spite of adequate liver vitamin A stores. A study was conducted to explore the effects of zinc deficiency on the metabolism of retinol-binding protein (RBP), the plasma transport protein for vitamin A. The plasma RBP level of zinc-deficient rats was only 14 ± 2 μg per milliliter In contrast, the plasma RBP level was 50 ± 2 μg per milliliter in control rats fed ad libitum on a zinc-sufficient diet and 36 ± 1 μg per milliliter in a zinc-sufficient group pair-fed with the zinc-deficient rats. Moreover, the liver RBP levels of the zinc-deficient group were only about 55 to 60 per cent of the levels of either the ad libitum or pair-fed control animals. The data suggest that zinc deficiency interferes with the synthesis of RBP by the liver and probably with the synthesis of some other plasma proteins as well. The low plasma vitamin A levels seen in zinc deficiency can be attributed to an impaired ability of the deficient rat to mobilize vitamin A from its liver in the form of the retinol-RBP complex.

Amino acid metabolism of experimental granulation tissue in vitro.

1. The intracellular volume in granulation tissue was about 15% of the total urea space. 2. The experimental granuloma has a greater ability to retain amino acids during the proliferation phase than later during the synthesis of collagen. 3. The synthesis of collagen and other proteins by granulation tissue is related to the concentrations of proline and glutamic acid in the medium. 4. The rate of synthesis of proline from glutamic acid in granulation-tissue slices is greatest during collagen synthesis. It is enhanced by lactate. 5. Extracellular cations influence the synthesis of collagen and ouabain is inhibitory. Synthesis of other proteins is less sensitive in this respect. 6. It is suggested that the synthesis of collagen is related to the supply of certain amino acids, especially proline, and hence to the redox balance, and also to the function of the cell wall.

Effects of the parasitoid, microplitis croceipes on the haemolymph proteins of the corn earworm, Heliothis zea

1. 1. Parasitism of the corn earworm, Heliothis zea, by Microplitis croceipes, resulted in differences in the number and location of host proteins when separated by disc electrophoresis. 2. 2. These results suggest that the parasitoid may break down or utilize certain host proteins and cause the release or synthesis of other proteins by the host. 3. 3. Additionally, the parasite may utilize or destroy certain host haemolymph proteins while causing a shift in free amino acids of the shot to the bound state or the secretion of other proteins into the haemolymph. 4. 4. The result demonstrates that the parasite extracts or uses a large amount of conjugated protein from the host during its development, while other organic constituents such as lipids appear not to be consumed as fast during the host-parasitoid association.

Properties of two nuclease genes in Neurospora crassa.

Two genes, nuc-1 and nuc-2, of Neurospora crassa which were responsible for the nucleic acid digestion, were located on linkage group 1 and 2, respectively. A temperature-sensitive mutant (B1ts-2) was obtained from a nuc-2 mutant. Nuclease mutants showed a reduced activity of nuclease N(3) which was found to be a complex consisting of nuclease N(3) (') and inhibitor molecule. Nuclease N(3), nuclease N(3) (') and inhibitor were partially purified and estimated to have the approximate molecular weights of 38,000, 12,500 and 25,000 respectively. A nuc-1 mutant produced the nuclease N(3) (') altered in thermolability. A nuc-2 mutant and B1ts-2 produced the inhibitor altered in the capacity to inhibit nuclease N(3) (') activity. The inhibitor prepared from B1ts-2 was more thermostable than that from other strains. From these results, it was suggested that the nuc-1 gene is the structural gene for nuclease N(3) (') and the nuc-2 gene that for the inhibitor. A possible involvement of this enzyme-inhibitor complex in the regulation of nuclease activity and synthesis of other proteins was suggested.

Quantitative studies of prococoonase synthesis and accumulation during development

Methods are described for the measurement of prococoonase in galeae at levels of the order of 0.05 μg per galea or higher, and for determination of the rate of zymogen synthesis, relative to synthesis of other proteins, at levels higher than 0.01%. These methods permit a biochemical description of differentiation in the galeae. Massive synthesis of prococoonase begins sometime between stages 8 II and 9 II, accelerates during subsequent stages, and eventually declines after reaching a level of more than 66% of total protein synthesis (10 10 molecules per cell per day) on stage 14 III. Accumulation of zymogen proceeds to a level of 45 μg per galea (3 to 6 × 10 10 molecules per cell) on stage 15 III, when extrusion from the cells begins. Although the rate of synthesis of prococoonase prior to turning on cannot be estimated exactly, it is at least four orders of magnitude lower than maximal. An early processing of prococoonase has been detected, involving a change of approximately 300 daltons in the apparent molecular weight. The time course of this processing suggests that it might be implicated in the process of secretion into the storage vacuole.

Cell division and collagen synthesis in cultured fibroblasts

Summary The rate of synthesis of collagen and of non-collagen proteins at high and at low cell density was determined in the established mouse embryo fibroblast line 3T6 and in several human fibroblast lines. Collagen synthesis is always more rapid in proliferating than in stationary cultures. In 3T6 and in “old” but not in “young” human fibroblast lines the percentage of collagen to total protein synthesized is higher in stationary than in proliferating cultures, not because collagen synthesis is stimulated at high cell density but because synthesis of other proteins is more repressed than collagen synthesis. Arresting cell proliferation by exposure of the cultures to thymidine does not change the rate of collagen synthesis relative to the synthesis of other proteins. Omission of serum from the culture medium strongly diminishes cell proliferation, DNA and protein synthesis. In growing 3T6 and “old” human fibroblast lines, omission of serum inhibits non-collagen protein synthesis more strongly than collagen synthesis, so that a higher percentage of the protein synthesized is collagen. No such effect is found in “young” human fibroblast lines.

Adaptation fonctionnelle des t-RNA à la biosynthèse protéique dans un système cellulaire hautement différencié: IV — Evolution des t-RNA dans la glande séricigène de Bombyx mori L. au cours du dernier âge larvaire

Summary The quantitative changes in the t-RNA species extracted from the post-ribosomal supernatant of the posterior part and of the middle part of the silkgland of Bombyx mori L during the growth phase of the gland and the phase of fibroin and sericin secretion of the 5 th larval instar have been studied. In the posterior part which produces fibroin, the level of t-RNA is increasing 10-fold from the 1 st to the 8 th day and rapidly decreasing during the two days before spinning. In the middle part, which produces sericin similar changes (a continuous 5-fold increase from the 1 st to the 7 th day) are shown. The quantitative changes in each of 17 t-RNA species were followed by in vitro heterologous acylation under optimum conditions. The t-RNA corresponding to the major amino acids of the silk proteins were predominant: t-RNA Ala , t-RNA Gly , t-RNA Ser and t-RNA Tyr in the posterior part, t-RNA Ser , t-RNA Ala , t-RNA Gly and t-RNA Asp in the middle part, account for 2/3 of the total supernatant t-RNA pools at the 8 th day and at the 7 th day respectively. Two groups of t-RNA can be distinguished. Those which have a role in fibroin production, have a maximum concentration at 7–8 days, and those which are primarly involved in the synthesis of other proteins, which reach a maximum around 4–5 days.

Effects of the parasitoid, Cardiochiles nigriceps, on the growth, development, and tissues of Heliothis virescens

Parasitism of the tobacco budworm, Heliothis virescens, by Cardiochiles nigriceps resulted in a reduction in weight gain by parasitized larvae. Host larvae parasitized during the second or fourth instars developed through the same number of moulting cycles as non-parasitized controls although the head capsule diameter of parasitized larvae was reduced. Both parasitized and control larvae entered the pharate pupal phase at the same time, but the parasitized larvae failed to shed the larval cuticle. When second instar host larvae were parasitized, the parasite emerged in 12 days, 1 day after the controls pupated. Parasites from larvae parasitized during the fourth instar required 11 days for development and emerged 3 days after the controls pupated. The specific gravity of the host haemolymph increased following parasitism. Differences in the number and location of host proteins separated by disk electrophoresis between parasitized and non-parasitized hosts occurred, suggesting the breakdown or utilization of certain host proteins and the release or synthesis of other proteins. Changes in the developing fat body of nonparasitized larvae were observed during the pharate pupal phase which resulted in the deposition of protein globules and enlargement of the fat body. The fat body of parasitized larvae did not change during the pharate pupal phase. Parasite larvae fed exclusively on haemolymph until 2 or 3 days before emergence, when the fat body was attacked.

Female specific protein: biosynthesis controlled by corpus allatum in Leucophaea maderae.

A specific protein is Present in serums of all of Leucophaea maderae that mature eggs but not in serums of males, nymphs of either sex, or females that are reproductively inactive. Ablations, microsurgery and remplantation showed that this protein is produced de novo under the influence of the corpus allatum hormone. This protein, essential for egg maturation, is synthesized in isolated abdimens of allatectomized females treated with 0.08 to 0.12 microgram of the t,t,t-isomer of the authentic juvenile hormone. The crops allatum hormone also stimulates toa similar degree the synthesis of other proteins which appear to be essential for egg maturation as well; these are not specific to females.

In Vitro Studies of Immunological Responses of Lymphoid Cells

In vitro studies of antibody synthesis have great attraction because they offer the hope of isolating and examining separately the variables involved in the interaction of antigen with lymphoid cells, which results in an immunological response. The chapter reviews such studies and critically analyzes the different experimental conditions employed, the types of data obtained, and their interpretation. The mechanisms and problems involved in antibody synthesis appear to be similar to those involved in synthesis of other proteins. Of more peculiarly, immunological interest are attempts to induce and demonstrate specific responses in vitro . One of the simplest changes to measure is increased DNA synthesis and that is applicable to stimulation by cell surface antigens. The phenomenon is clear enough, but despite many ingenious experiments, this chapter shows how little is yet understand about its mechanism. The advantages of an in vitro analysis of the immunological response were clear from the earliest times. In the study of a mechanism involving a complex series of events and the possible interaction of cell populations, it is essential to be able to identify the cells under study, regulate the conditions of their exposure to antigen, and to study their subsequent morphological and biochemical response. In spite of these potential advantages, there was a long induction period during which a great deal of effort was expended and relatively little achieved.

The nature of polysomes isolated from spleen cells of rats stimulated by antigen.

Kern an(d co-workers1 were the first to examine the molecular mechanism of imnmunoglobulin synthesis and concluded that this process is similar to the synthesis of other proteins. Recently, lymphoid tissues have been studied by more refined electron microscopic2' and biochemical techniques4-6 in an effort to examine details of the mechanism of synthesis of antibody. The data presented in many of these studies are difficult to reconcile with present models of polypeptide synthesis on polysomes. The current hypothesis is that the size of a fully functional polysome should be compatible with the size of the product of synthesis.7 Thus for L and H chains one would expect polysomes in the range of 7 and 15 ribosomes, respectively. The examination of polysomal units of immunoglobulin synthesis isolated from lymph node and spleen homogenates has been complicated by the presence of highactivity ribonucleases (RNases). These tissues seem to lack the RNase inhibitors present in liver cells.8 The contradictory results of experiments in this field might be due to endogenous RNases. On the other hand, synthesis of an antibody molecule might, proceed differently from the model proposed for other polypeptides. The work reported here is the result of efforts to develop a system in which biochemical and morphological studies could be made of polysomes of lymphoid tissue cells during response to antigen injection. The first objective was to devise a means to reduce the degradative action of endonucleases on the preparations being studied. The initial effort to develop such a system was partially successful.9 The experiments presented here indicate success in devising a method to block RNase action completely in homogenates of spleen and lymph nodes. Interpretation of the results of these experiments suggests that some discrepancies may exist between accepted models of protein synthesis and the types of polysomes involved in the assembly of L and H chains of antibody globulins. Materials and Methods.-Animals and immunization: Sprague-Dawley male albino rats weighing between 150 and 300 gm were used in all experiments. They were immunized in two ways: primary immunization consisted of an intravenous injection of 1.0 ml of Salmonella typhi formalinkilled vaccine;10 hyperimmunized animals were given three intravenous injections of 1.0 mnl of the vaccine on 3 consecutive days and 30 days later received a fourth intravenous injection to serve as a booster. No differences were noted in the results of experiments using rats given either immunizing treatment, and therefore no distinction will be made between hyperimmunized and nonhyperimmunized animals. Isotopes: Reconstituted, C'4-labeled Chlorella hydrolysate prepared by Schwarz BioResearch was used in all experiments. Cell preparation, incubation, and centrifugation: Rats were killed by exsanguination under ether anesthesia; the spleen was rapidly removed, placed in ice-cold buffered special saline (BSS, 0.03 M tris, 0.14 M NaCl, 0.005 M KCI, 0.0015 M MgC92), and finely minced. Splenic fragments were then forced gently through an 80-mesh stainless-steel screen by means of a syringe plunger, with frequent BSS washings. Collected cells were washed twice by centrifugation at 250 X g in cold BSS with e-thylenediaminetetraacetate (EDTA) and then in cold BSS without EDTA. The cell suspension from one spleen was then incubated in a Dounce homogenizer vessel in 2.0 ml of Eagle's

Factors in the Stimulation of Protein Synthesis by Subcellular Preparations from Rat Liver

Abstract The control of protein synthesis has been investigated in subcellular systems from livers of rats with plasma protein overproduction following nephrotoxic serum. The following observations were made. The microsomal fraction from nephrotic liver exhibited an increased synthesis of acid-insoluble protein and of plasma albumin, as measured by amino acid incorporation per mg of ribonucleic acid present. The synthesis of plasma albumin showed a much greater increase than did the synthesis of other proteins. This is in contrast to the situation in regenerating liver, in which the increase in plasma albumin synthesis was of the same order of magnitude as the increase in acid-insoluble protein. Ribosomes or polysomes, prepared from nephrotic liver with deoxycholate, did not differ from normal liver in their ability to synthesize protein. This finding, together with the observation that mixtures of control and nephrotic microsomal fractions behaved like the nephrotic system, suggested that the difference between control and nephrotic microsomes was not the result of any inherent difference in ribosomes but might be due to the presence of additional amounts of messenger RNA. Polysomes prepared from nephrotic liver without the use of deoxycholate but still containing small amounts of attached membrane protein showed slight but significant increases in plasma albumin synthesis, averaging 33%. Cell sap from nephrotic liver was more active than that from controls in promoting protein and plasma albumin synthesis by microsomes. This difference was ascribed to the presence of increased amounts of messenger RNA in this fraction on the basis of indirect evidence.

Action de la 8-azaguanine sur la synthèse de pénicillinase chez Bacillus cereus

Action of 8-azaguanine on the synthesis of penicillinase in Bacillus cereus Previous work on Bacillus cereus has indicated that protein synthesis is partially inhibited in the presence of azaguanine and that the synthesis of penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) is apparently more affected by the analog than the synthesis of other proteins. A search for the presence of an “abnormal” penicillinase under these conditions was made in the present work by immunological reactions and by column chromatography. Among the proteins secreted by the bacteria in presence of azaguanine, there is no other protein similar enough to penicillinase to be detected by immunological reactions. Similarly, the column chromatography experiments do not favour the hypothesis that other, different proteins might be synthesized under these conditions.

The role of ascorbic acid in the biosynthesis of collagen I. Ascorbic acid requirement by embryonic chick tibia in tissue culture

Embryonic chick tibias, when incubated in a chemically defined medium, grow and synthesize collagen for several days. The tissues display a marked dependence upon the presence of L-ascorbic acid in the culture medium for normal growth. Deletion of ascorbic acid reduces collagen synthesis by the bones after the first 2 days in culture. A less drastic reduction in the synthesis of other proteins, DNA and RNA, was also observed. The rate of synthesis of these substances was rapidly restored by the addition of ascorbic acid.

Cell-free protein synthesis directed by coliphage MS2 RNA: Synthesis of intact viral coat protein and other products

Coliphage MS2 RNA directs the synthesis in Escherichia coli extracts of a protein product the tryptic peptides of which correspond to the peptides of the phage coat protein. Fingerprint analysis of the synthetic protein labeled with various [14C]amino aeids indicated that in each case only those coat protein peptides which contain a particular amino acid were labeled. The formation of whole coat protein molecules as the predominant product was shown by co-chromatography on Sephadex with authentic phage coat subunit. Sequential synthesis of coat protein ending at the carboxyl end was observed by pulse-labeling with radioactive amino acid and by analysis of the peptides formed in the presence of puromycin. In addition to phage coat protein, MS2 RNA directed the synthesis of other proteins. These could be preferentially labeled with histidine, an amino acid lacking in the coat protein, and partially separated on Sephadex.

Hemin synthesis in the developing frog embryo and its stimulation by frog liver RNA

Hemin was synthesized from δ-aminolevulinic acid-4-C 14 by intact and minced frog embryos; the isotopic activity of the hemin samples is a measure of the activity of the enzymes of this pathway of porphyrin biosynthesis. Administration of the isotopic hemin precursor to intact embryos revealed low levels of synthesis until stage 25, but this is attributed to the poor penetration of δ-aminolevulinic acid into the embryos due to their relatively impermeable surface coat. Cutting each embryo into 4 or 5 pieces (mincing) allowed penetration of the hemin precursor into the embryo. It appears there is a synthesis of the enzymes of this porphyrin biosynthetic pathway in the minced embryos, since puromycin inhibited the incorporation of labeled amino acids into protein, and also the incorporation of δ-aminolevulinic acid-4-C 14 into hemin of minced stage 20 embryos. The minced embryos showed a slight increase in hemin synthesis from gastrulation to neurulation and a larger increase to the tailbud stage. The level of hemin formation in minced stage 25 larvae was lower than that of intact stage 25 larvae; at the feeding larval stage the labeled compound can enter the organism and is converted to hemin more efficiently than by minced stage 25 larvae. The real course of hemin synthesis in developing frog embryos, as revealed from the results obtained with both minced and intact embryos, is that of a gradual increase from gastrulation to neurulation, followed by an increased rate of hemin synthesis at tailbud and larval stages. The addition of frog liver RNA (1 mg/ml) to minced stage 20 embryos consistently stimulated the level of hemin synthesis above that of untreated minced embryos. Frog liver DNA and RNA of frog spleens, muscle, and red blood cells, as well as mouse ascites tumor cell RNA (1 mg/ml), did not stimulate hemin synthesis. Stimulation is also evoked by a mixture of the four ribonucleosides. Puromycin inhibits both protein and hemin synthesis of minced embryos while liver RNA stimulates the incorporation of labeled amino acids into the protein fraction. It appears that hemin synthesis is linked to the synthesis of the enzymes of porphyrin biosynthesis in these experiments. Phenol extraction of RNA from centrifugal fractions of liver homogenates revealed that there is active RNA in the ultracentrifugal supernatant fraction and that a transfer RNA fraction isolated from the ultracentrifugal supernatant of frog liver retains the ability to stimulate hemin synthesis of minced stage 20 embryos. Hot phenol extracts of the interphase of cold phenol extracts of frog liver also stimulated hemin synthesis of minced gastrulae cultured for 24 hours, but the stimulation by the four ribonucleosides and the RNA of the ultracentrifugal supernatant argue against an effect of messenger-RNA molecules. It is likely that supplying precursors of RNA synthesis, or transfer RNA, to cut frog embryos can stimulate the synthesis of enzymes synthesizing hemin, as well as the synthesis of other proteins.