Control Of Protein Synthesis Research Articles

Analysis of lens protein synthesis in a cataractous mutant mouse: The Cat Fraser

The cataract produced by the dominant Cat Fraser gene in mouse is associated with quantitative changes in lens proteins (crystallin) and with capsule abnormalities. We have analyzed and compared the protein synthesis in control and mutant lenses using [ 3H]leucine and [ 3H]proline incorporation. The specific activities of free [ 3H]leucine in the intracellular pools of the two mouse strains were identical, while the incorporation of both labelled amino acids in proteins was largely increased in Cat Fraser lens. These data indicate that the higher labelling of Cat Fraser lens proteins reflects a true change in the cellular synthesis activity by Cat Fraser lens cells. Despite the enhanced type IV collagen synthesis by Cat Fraser epithelial cells, the amount of type IV collagen in Cat Fraser capsule is lower than in control. This altered type-IV collagen metabolism may disturb the structure of Cat Fraser capsule which becomes thicker.

The effect of juvenile hormone on protein synthesis in the transparent accessory gland of male Rhodnius prolixus

Feeding stimulates the accumulation of protein by the transparent accessory reproductive glands (TARG), allatectomy abolishes the increase, and severing the nervous connections between the corpus allatum (CA) and the brain raises the level of accumulation after feeding. TARG removed from allatectomised females exhibit a markedly decreased level of incorporation of [ 3H]-leucine into protein in vitro, and topical application of juvenile hormone (JH) to allatectomised males restores the ability of the glands to incorporate [ 3H]-leucine in vitro. Severing the nerves between the CA and the brain increases the rate of incorporation in vitro, but in both operated and normal males, the rate of incorporation decreases with time after feeding. JH applied topically to intact animals during the falling phase of incorporation, or applied directly to TARG in vitro from such animals, stimulates incorporation. These data indicate that JH from the CA controls protein synthesis in the TARG and that the CA in the male of Rhodnius may be subject to different controls than in the female.

Enhancement of the Cytotoxic Effect of Anti-Carcinoembryonic Antigen Immunotoxins by Adenovirus and Carboxylic lonophores<xref ref-type="fn" rid="FN1">2</xref>

Immunotoxins (monoclonal antibodies-ricin A-chain conjugates) directed against the tumor-associated antigen carcinoembryonic antigen (CEA) are selective in vitro cytotoxins for human adenocarcinoma cells. However, the kinetics of intoxication are relatively slow. The effects of the UV radiation-inactivated human adenovirus and the carboxylic ionophores monensin and nigericin were examined on immunotoxin cytotoxicity to the human colorectal adenocarcinoma cell line LoVo. In a 16-hour cytotoxicity assay, adenovirus reduced 33-fold the median inhibitory concentration from 3 X 10(-8) M to 9 X 10(-10) M. In timed cytotoxicity assays, 50% of control protein synthesis was reached in immunotoxin-treated cells twentyfold faster in the presence of adenovirus (0.5 hr) than in its absence (10 hr). Adenovirus produced no enhancement of immunotoxin effect on a control cell line or on a control immunotoxin on LoVo cells, demonstrating specificity of adenovirus effect. In addition, specific immunotoxin constructed with a nonreducible thioether bond, alone or with adenovirus, produced no toxicity on LoVo cells. These results suggest that both cell surface binding and presence of a reducible disulfide bond in the conjugate are necessary for adenovirus effect. Similar potentiation of the cytotoxicity of anti-CEA immunotoxins was produced by monensin and nigericin. These studies demonstrate that both adenovirus and carboxylic ionophores are potentiators of immunotoxins directed against the CEA, producing cytotoxicity equivalent to that of ricin but specific for CEA-positive adenocarcinoma cells in culture.

Early hypomethylation of 2'-O-ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCl4.

Carbon tetrachloride (CCl4) treatment of rats produces an early defect in methylation of hepatocyte ribosomal RNA, which occurs concurrently with a defect in the protein synthetic capacity of isolated ribosomes. The CCl4-induced methylation defect is specific for the 2'-O-ribose position, and a corresponding proportional increase in m7G base methylation occurs in vivo. Undermethylated ribosomal subunits (rRNA) from CCl4-treated preparations can be methylated in vitro to a much greater extent than those from control preparations, and in vitro methylation restores their functional capacity. In vitro methylation of treated ribosomal subunits (which restores functional capacity) occurs at 2'-O-ribose positions (largely G residues). In contrast, in vitro methylation of control ribosomal subunits (which does not affect functional activity) represents base methylation as m7G, sites which are apparently methylated in treated preparations in vivo. Methylation/demethylation of 2'-O-ribose sites in rRNA exposed on the surface of cytoplasmic ribosomal subunits may represent an important cellular mechanism for controlling protein synthesis in quiescent hepatocytes, and it appears that CCl4 disrupts protein synthesis by inhibiting this 2'-O-ribose methylation.

Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [(3)H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [(14)C]leucine into protein and respiration as measured by O(2) consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O(2) consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.

Involvement of the membrane skeleton in the regulation of the cAMP-independent protein kinase and a protein phosphatase that control protein synthesis.

The ultimate targets of transformational changes in cells are the sites in the nucleus and cytoplasm that affect transcriptional and translational control of protein synthesis. Products of a number of oncogenes appear to be analogues of proteins involved in the steps of transmembrane signalling (hormones or growth factors, receptors, G-type regulatory proteins, and tyrosine kinases), and some insight into the specific mechanism is emerging (for review see [1]). However, little is known of the mechanism by which a signal is transmitted from the inner surface of the plasma membrane to specific targets in the nucleus and cytoplasm. In at least some cases, changes in the activity of specific cyclic adenosine monophosphate (cAMP)-independent protein kinases and possibly phosphoprotein phosphatases appear to be involved.

A (re)initiation‐dependent cell‐free protein‐synthesis system from mouse erythroleukemia cells

Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis. When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post-mitochondrial supernatants show a high in vitro protein-synthesis activity. Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine. Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells. This reduction of the protein-synthesizing capacity is also observed in vivo. Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non-globin protein synthesis is reduced and globin is preferentially synthesized. Haemin (40 microM) as well as purified eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin-controlled eIF-2 alpha kinase. This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.

Role of heme and iron metabolism in controlling protein synthesis.

Journal of the American Geriatrics SocietyVolume 34, Issue 8 p. 593-600 Role of Heme and Iron Metabolism in Controlling Protein Synthesis Dr. David L. Marcus PhD, Corresponding Author Dr. David L. Marcus PhDDivision of Geriatrics, Department of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016.Search for more papers by this authorMichael L. Freedman MD, Michael L. Freedman MDSearch for more papers by this author Dr. David L. Marcus PhD, Corresponding Author Dr. David L. Marcus PhDDivision of Geriatrics, Department of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016.Search for more papers by this authorMichael L. Freedman MD, Michael L. Freedman MDSearch for more papers by this author First published: August 1986 https://doi.org/10.1111/j.1532-5415.1986.tb05765.xCitations: 2 From the Division of Geriatrics, Department of Medicine, New York University Medical Center, New York, New York. Supported in part by a grant from the Metropolitan Jewish Geriatric Center of New York. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume34, Issue8August 1986Pages 593-600 RelatedInformation

Intracellular pH controls growth factor-induced ribosomal protein S6 phosphorylation and protein synthesis in the G0→G1 transition of fibroblasts

Mitogen-induced intracellular alkalinization mediated by activation of a Na +/H + antiporter is a common feature of eukaryotic cells stimulated to divide. A Chinese hamster fibroblast mutant (PS120) lacking Na +/H + antiport activity (Pouysségur et al., Proc natl acad sci US 81 (1984) 4833) [42] possesses an intracellular pH (pH i ) 0.2–0.3 units lower than the wild type (CCL39) and requires a more alkaline pHout (pH o ) for growth. Here, we show that serum-stimulated ribosomal protein S6 phosphorylation, protein synthesis activation and DNA synthesis re-initiation are pH-regulated events that display a similar threshold pH o value (6.60) in CCL39 cells. pH-Dependencies for initiation of all three events are shifted toward higher pH o values in the mutant PS120, indicating that growth factor-induced alkalinization has a permissive effect on the pleiotypic response. However, cytoplasmic alkalinization per se is insufficient to trigger S6 phosphorylation, polysome formation, and subsequent DNA synthesis. Transient exposure to a non-permissive pH o (6.5) inhibits both the rate of leucine incorporation into proteins and the progression through the G1 phase of the cell cycle. In contrast, cells committed to DNA synthesis are unaltered by the acidic pH o . These observations suggest that pH i by controlling the rate of protein synthesis play a determinant role in the control of cell division.

Modelling protein synthesis, a step to an accurate estimate of net primary production: Phaeocystis pouchetii colonies in Belgian coastal waters

Light and dark rates of net protein synthesis by Phaeocystis pouchetii colonies were measured in Belgian coastal waters during the spring bloom of 1984 by following I4C-bicarbonate and l5N03/13NH4: assimilation into cellular proteins during natural 1ight:dark cycles. A close agreement was found between the 2 tracer methods. Ambient inorganic nitrogen was shown to be the most important factor controlling protein synthesis in the light, which was considered to proceed at a constant rate in the whole euphotic layer. Protein synthesis in the dark, on the other hand, was found to be controlled by both inorganic N and 'previous light history' of the cells. The relation between specific protein synthesis and external inorganic nitrogen obeyed Michaelis-Menten kinetics for both light and dark processes. The Michaelis-Menten constant K, was 4 µmoles N 1-1 while the maximum specific protein synthesis rates p,, were respectively 3 and 1.8 % h-' for the light and dark processes. The relation between protein assimilation in the dark and 'previous light history' could be expressed by a linear relation between dark specific protein synthesis and the number of hours spent at 3 8 J cm-'h-', the energy threshold for the occurrence of dark protein synthesis. From the knowledge of these control mechanisms a simple mathematical model was established in order to calculate daily integrated net primary production from the hourly variations of light intensity in the water column and ambient inorganic N concentrations. These estimates have been compared with those calculated from classical methods for measurements of primary production.

Lymphocyte blastogenesis. Post-transcriptional controls of protein synthesis

To better understand the regulation of macromolecular synthesis in the response of lymphocytes to a mitogen, we have used two-dimensional electrophoresis to search for specificity in the early increase seen in protein synthesis in human lymphocytes treated with phytohemagglutinin and examined the role of new RNA synthesis in this response. Our results confirm a major increase in overall protein synthesis after 4 h of phytohemagglutinin treatment. A further disproportionate increase in the synthetic rates of certain polypeptides was observed using two-dimensional polyacrylamide gel electrophoresis. While actinomycin-D reduced protein synthesis to a level below that of untreated cells, phytohemagglutinin nonetheless enhanced total protein synthesis even in the presence of actinomycin. Some, but not all, of the disproportionate increases in synthesis seen for certain polypeptides are blocked by actinomycin. These results imply the existence of multiple mechanisms controlling protein synthesis early in the course of lymphocyte stimulation. At least some of these do not require new RNA synthesis and thus operate at a post-transcriptional level.

Aortic perfusion pressure as a determinant of cardiac protein synthesis.

Mechanical parameters and intracellular mediators that may control protein synthesis have been studied in isolated rat hearts subjected to increased aortic pressure or induced to perform cardiac work. Elevation of aortic pressure from 60 to 120 mmHg in Langendorff preparations with glucose, glucose plus insulin, or pyruvate raised the rate of protein synthesis during the 2nd h of perfusion. These effects involved faster rates of both peptide chain initiation and elongation. In working hearts supplied glucose or glucose plus insulin, higher rate of synthesis were observed in both the 1st and 2nd h of perfusion, compared with Langendorff preparations perfused at 60 mmHg. Intracellular levels of glucose 6-phosphate, ATP/ADP ratio, adenylate energy charge, or creatine phosphate/creatine did not correlate with the rate of protein synthesis in beating control hearts. When ventricular pressure development was prevented by ventricular draining and hearts were arrested with tetrodotoxin, protein synthesis still increased as a function of perfusion pressure. Oxygen consumption increased as aortic pressure was raised in beating-drained hearts but was unaffected in arrested-drained hearts. These results indicate that intraventricular pressure development, cardiac contraction, oxygen consumption, glucose 6-phosphate, energy availability, and coronary flow could be dissociated from the stimulatory effect of higher aortic pressures on protein synthesis and suggested that stretch of the ventricular wall, as a consequence of increased aortic pressure, could be the mechanical parameter most closely related to the increase in protein synthesis.

Internal pH of Xenopus oocytes: A study of the mechanism and role of pH changes during meiotic maturation

The internal pH (pH i) of Xenopus laevis oocytes, as measured by the DMO method, covered a broad range of values from 7.06 ± 0.01 to 7.93 ± 0.01, with a mean value of 7.43 ± 0.03. The pH i measured by DMO and microelectrodes was nearly identical in control and maturing oocytes from the same batch. The oocytes from most females elevated their pH i in response to progesterone, reaching a maximum elevation of 0.30 ± 0.03 pH units above control values at 100% germinal vesicle breakdown (GVBD). However, some females were found to contain oocytes that already had an elevated pH i of 7.71 ± 0.03 which did not significantly increase during maturation. Human chorionic gonadotrophin (hcG)-stimulated females had oocytes with slightly higher control pH i values than oocytes from nonstimulated females but still showed the same elevation in response to progesterone. Thus, the “stimulated” state of oocyte physiology as induced by hcG did not account for the variation in control pH i and responsiveness to progesterone. Other aspects of this variability are discussed. Elevating or lowering the external pH is shown to elevate and lower pH i, respectively, in a stable and predictable manner. Using this approach to change pH i we have found no effect of changes in pH i on the rate of protein synthesis in control and maturing oocytes. Similarly, pH i had only a slight facilitating effect on the rate of GVBD. A pH indicator gel was used to demonstrate that the pH i increase during oocyte maturation involved an acid efflux. We conclude that an elevated pH i is not necessary for oocyte maturation, yet the mechanism of the pH i elevation is discussed as a possible lead to events that are necessary.

Diurnal regulation of translational capacity in a cell‐free system from rat liver

Abstract Polysomal messenger RNA from rat liver microsomal fractions was translated in a hepatic in vitro system containing among other components Sephadex G‐25 eluate. 3H leucine incorporation was measured both in overall protein and in integral membrane proteins prepared from microsomal membranes by treatment with EDTA and 2M LiCl. In the first series of experiments, microsomes from different phases of the diurnal cycle were incubated for protein synthesis with the same G‐25 eluate from a single time of day. The rates of protein synthesis revealed a diurnal rhythmicity with major maximum at 300h. In the second series, two microsomal fractions from different diurnal phases were pre‐incubated with their own post‐microsomal supernatant and with that from another time of day. The incubation of the re‐isolated microsomes was carried out with two G‐25 eluates from different phases. These experiments indicated the existence of a diurnally fluctuating cytosolic factor or factors controlling protein synthesis. T...

The role of the maturation-promoting factor in controlling protein synthesis in Xenopus oocytes

Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.

Influence of diabetes control on synthesis of protein and basement membrane collagen in isolated glomeruli of diabetic rats.

Diabetic rats were treated with insulin at various dosages and for various periods of time. The influence of metabolic control on the synthesis of protein and basement membrane collagen of isolated glomeruli was investigated. The protein and basement membrane collagen synthesis was increased in the untreated diabetic rats compared to non-diabetic controls. The synthesis was not affected by high doses of insulin with brief normalization of the blood sugar. Insulin treatment from the beginning of diabetes only led to a normalization of protein synthesis in moderate metabolic control. On the other hand, a rise of the basement membrane collagen synthesis could only be prevented by a strict metabolic control of the rats. The results show that basement membrane collagen synthesis reacts very sensitively to the diabetic metabolic situation. Insulin deficiency itself does not appear to be alone responsible for alterations of basement membrane synthesis in diabetes.

Effects of diabetes on protein turnover in cardiac muscle.

Effects of alloxan diabetes of 10-day duration on protein turnover were investigated in hearts perfused with buffers simulating control and diabetic sera. Diabetes produced a 30% inhibition of protein synthesis in hearts perfused as Langendorff or working preparations. This reduction was attributable to a 20% fall in RNA concentration and a 10% decrease in efficiency of protein synthesis. Determination of RNA in ribosomal subunits indicated that the reduction in efficiency that was observed with diabetes may be due to an inhibition of polypeptide chain elongation/termination. Pharmacological levels of insulin (25 mU/ml) and cardiac work stimulated protein synthesis in both control and diabetic hearts. Effects of diabetes and insulin on protein synthesis in isolated heart muscle cells were similar to those found in whole heart. Diabetes increased protein degradation in hearts perfused with buffer similating diabetic serum and under conditions of cardiac work. Insulin (25 mU/ml) decreased protein degradation in both control and diabetic hearts. These studies indicate that long-term diabetes produces a greater negative nitrogen balance that, in contrast to control hearts, cannot be normalized by pharmacological levels of insulin or by cardiac work.

Globin synthesis and erythroid differentiation in a Friend cell variant deficient in heme synthesis.

Friend erythroleukemia cells of line Fw are noninducible for hemoglobin synthesis by dimethyl sulfoxide, butyric acid, and other agents. However, these agents were found to induce hemoglobin synthesis if the cells were also treated with exogenous hemin. Butyric acid (or, to a lesser extent, dimethyl sulfoxide) by itself induced accumulation of the erythroid-specific membrane protein spectrin. The basis of the control of globin gene expression by hemin was investigated. Hemin does not control globin and total protein synthesis via the action of the hemin-controlled repressor. Rather, hemin, alone or in combination with dimethyl sulfoxide, induces accumulation of globin mRNA and nuclear globin RNA sequences. Direct measurements of heme synthesis indicate that Fw cells may be significantly deficient in heme metabolism.

Intracellular pH controls protein synthesis rate in the sea urchin egg and early embryo

Direct comparisons between intracellular pH and protein synthesis in the sea urchin egg and early embryo show that pH controls protein synthesis rate in a highly sensitive and reversible manner. The entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in intracellular pH. However, unfertilized eggs whose intracellular pH has been raised artificially by ammonia take at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. This time lag for ammonia activation and the decrease in protein synthesis rate during mitosis suggest that other unknown factors can also influence protein synthesis rate during fertilization and early embryogenesis.

Effects of testosterone on messenger ribonucleic acid and protein synthesis in rat seminal vesicle.

In a previous report [Higgins et al. (1976) Biochem. J.158, 271-282] we described the effects of alterations in androgen status on the synthesis of two basic secretory proteins of the rat seminal vesicle. In the present paper we examine the effects of testosterone on the activity of mRNA in the seminal vesicle. Total cellular poly(A)-rich RNA was isolated and translated in a cell-free system prepared from wheat germ. Translation products were separated on denaturing polyacrylamide gels and the protein bands corresponding to the two basic secretory proteins were identified immunologically. Incorporation of radioactive methionine into these bands was taken as a measure of the individual mRNA activities. Total mRNA activity was estimated by radioactivity in total acid-precipitable material. The results show that 1 to 2 weeks after castration the activities of mRNA molecules for the basic secretory proteins were decreased 10-20-fold on a tissue basis. Testosterone given in vivo rapidly and substantially restores mRNA activity to normal. Since these changes correlate closely with variations in the rates of synthesis of the secretory proteins in whole cells it suggests that androgenic steroids control protein synthesis chiefly via mRNA availability. In this respect their action resembles those of other steroid hormones acting in other systems. However, these effects of testosterone on the mRNA molecules for the major secretory proteins could not be distinguished from those on total mRNA. Thus the proportion of the total mRNA population accounted for by the two specific mRNA molecules showed less than a 2-fold variation with androgen status. Similarly the two secretory proteins always accounted for 25-33% of general protein synthesis. This is in sharp contrast with the markedly differential effects of other steroid hormones controlling synthesis of major proteins in other well-studied systems. We interpret our results as indicating that testosterone regulates the mRNA population of the seminal vesicle as a whole.