The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats.

The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting. In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate. Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone. The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA. This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats. In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35%. In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22%. In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration. The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.     

Rates of growth and protein synthesis correlated with nucleic acid content in fry of rainbow trout, Oncorhynchus mykiss: effects of age and temperature

Groups of recently hatched fry of rainbow trout, Oncorhynchus mykiss were maintained in the laboratory in order to investigate the effects of age, ration level and temperature on whole body growth, nucleic acid concentrations, protein synthesis rates and enzyme activities. In fry of up to 30 days after hatching, which were feeding but still had some yolk sac, no significant change in mean RNA concentration was observed with ration level. In older fry of 50 days or more, when the yolk sac was completely absorbed and exogenous feeding fully established, the concentration of RNA was correlated with the rate of protein growth. RNA concentrations and activities of citrate synthase and lactate dehydrogenase were significantly different between fed and starved fry. As water temperature was raised (from 5 to 15° C), higher rates of protein growth were brought about by an increase in the rate of protein synthesis and also by increased efficiency of retention of synthesized protein (reduced protein turnover). In fed fry, no change in RNA concentration was found with increasing temperature, while the amount of RNA per cell (RNA: DNA) decreased, indicating that increased rates of protein synthesis were due to increased RNA efficiency.     

Effect of recombinant human tumour necrosis factor alpha on protein synthesis in liver, skeletal muscle and skin of rats.

Bacterial endotoxins cause enhanced protein metabolism in liver, and protein catabolism in muscle and skin. These effects may be mediated by cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF). The study investigates the timing and magnitude of effects of recombinant human TNF alpha on protein synthesis and protein and RNA content of the liver, tibialis muscle and skin of Wistar rats. Intravenous doses of 30 and 300 micrograms/kg of body weight were used and effects examined 8 h and 24 h after injection. Muscle protein content and synthetic rate were reduced at 8 h post-injection by over 18% and 20% respectively. Protein synthesis returned to normal after the lowest dose but remained depressed 24 h after the highest dose due to the accompanying anorexia. Opposite effects were observed in liver. Protein fractional synthetic rate (FSR) was increased by over 26% at 8 h post-injection and remained elevated 24 h after the higher but not lower dose of TNF. Total protein and RNA contents were significantly higher than controls at this time. Skin protein synthesis was unaffected by TNF; however an increase in protein and RNA content was observed at 8 h post-injection with the lower dose of TNF. Liver and muscle respond in a similar but more rapid way to TNF than to endotoxin. The response of skin is however totally different. While muscle may contribute amino acids for enhanced hepatic protein synthesis following exposure to TNF, skin does not.     

Thyroid hormones and muscle protein turnover. The effect of thyroid-hormone deficiency and replacement in thryoidectomized and hypophysectomized rats.

We have investigated the effects of thyroidectomy, hypophysectomy and 3,3',5-tri-iodothyronine replacement on protein synthesis and degradation in skeletal muscle in vivo. Thyroidectomy resulted in a decrease in the rate of protein synthesis as a result of a loss of RNA. However, RNA activity, the rate of protein synthesis per unit of RNA, was not decreased. This was the case in both young growing rats and mature nongrowing rats. Tri-iodothyronine treatment of thyroidectomized rats increased protein synthesis by increasing RNA concentration without changes in RNA activity, and this occurred even when food intake was restricted to prevent any increase in growth. The rate of protein degradation was decreased by thyroidectomy and increased by tri-iodo-thyronine replacement in both animals fed ad libitum and food-restricted animals. Hypophysectomy decreased protein synthesis by decreasing both RNA concentration and activity. these changes were reversed by tri-iodothyronine treatment even in the presence of persistent marked hypoinsulinaemia. This indicates that tri-iodothyronine can activate athe translational phase of protein synthesis in muscle in the absence of significant quantities of insulin. However, tri-iodothyronine does not seem to be obligatory for the maintenance of normal RNA activity in muscle, since in the thyroidectomized rat, in which plasma insulin concentrations are normal, RNA activity is maintained. From a consideration of the magnitude of changes in RNA activity observed in these experiments, it would appear that alterations in rates of elongation as well as initiation are involved in the changes in RNA activity.     

Chromosomal protein synthesis during erythropoiesis in the mouse spleen

Induced erythropoiesis in the mouse spleen was employed to study chromosomal protein synthesis during erythroid cell development. Splenic erythropoiesis occurring after phenylhydrazine induced hemolysis can be divided into an early phase during which nuclear RNA polymerase activity and RNA production are maximal and a late phase in which hemoglobin synthesis and DNA accumulation are maximal. Chromatin was isolated from splenic tissue during both the early and late phases of erythropoiesis as well as from non-anemic animals. The total protein content of chromatin from the early erythroid phase was greater than that of chromatin from the late erythroid phase or from non-anemic controls. The increase was due to a coordinate increase in the concentration of both histone and nonhistone proteins. During late erythropoiesis, the concentration of each returned to pre-anemic levels. Total histone synthesis increased 2.6-fold during early erythropoiesis as compared with the pre-anemic state and remained elevated in late erythropoiesis. The increase in histone synthesis was due to an increase in the synthesis of all five major histone proteins. Nonhistone protein synthesis was more active than that of histones in the pre-anemic spleen and rose only slightly during early erythropoiesis, returning to preanemic levels during late erythropoiesis. Fractionation of nonhistone proteins on SDS-urea polyacrylamide gels revealed complex patterns with significant differences between the pattern of erythroid spleen non-histone proteins and that of the pre-anemic spleen. Analysis of the incorporation of ³H-valine into the non-histone proteins indicated that during early erythropoiesis there was a generalized increase in nonhistone protein synthesis. During the late erythroid phase, the decline in non-histone protein synthesis was most marked for the higher molecular weight proteins.     

Turnover of muscle protein in the fowl. Changes in rates of protein synthesis and breakdown during hypertrophy of the anterior and posterior latissimus dorsi muscles.

Measurements were made of the growth and of the changes in rates of protein turnover in the anterior latissimus dorsi muscle of the adult fowl in response to the attachment of a weight to one wing. Over 58 days there was a 140% increase in the protein content with similar increases in the RNA and DNA contents. The fractional rate of protein synthesis, measured by the continuous-infusion technique using [14C]proline, increased markedly during hypertrophy. This increase was mediated initially (after 1 day) by an increase in the RNA activity but at all other times reflected the higher RNA content. The rate of protein degradation, calculated from the difference between the synthesis and growth rates, appeared to increase and remain elevated for at least 4 weeks. At no time was there any suggestion of a fall in the rate of degradation. The following events are discussed as central to the changes that occur during skeletal-muscle hypertrophy. 1. Nuclear proliferation is necessary to maintain the characteristic synthesis rate because of the inability of existing nuclei to 'manage' increased protein synthesis for more than a limited period. 2. The increased protein breakdown during hypertrophy is consistent with the known over-production of a new muscle fibres and may indicate some 'wastage' during the growth. Such wastage may also be associated with myofibrillar proliferation. 3. Muscle stretch must be recognized as the major activator of growth and as such can be compared with the 'pleiotypic activators' that have been described for cells in culture.     

Protein-carbohydrate interaction. A turbidimetric study of the interaction of concanavalin A with amylopectin and glycogen and some of their enzymic and chemical degradation products

1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase.     

The effect of ethylene on RNA and protein synthesis in germinating Manketti (Ricinodendron rautanenii Schinz) seeds

Amino acid uptake and protein synthesis were monitored in the embryonic axis, cotyledons and endosperm of manketti seeds (Ricinodendron rautanenii Schinz) during imbition, dormancy and dormancy-breaking by ethylene. Protein synthesis increased in all tissues within 48 h of imbibition and was strongly inhibited by cycloheximide and actinomycin D. No marked increase in protein synthesis was observed following ethylene treatment suggesting that qualitative rather than quantitative changes in protein synthesis were associated with dormancy-breaking. Seed germination was relatively insensitive to treatment with actinomycin D in marked contrast to the inhibition observed with cycloheximide.It is suggested that dormancy-breaking by ethylene may require protein synthesis but not DNA-dependent RNA synthesis.     

The relation between polyoma T-antigen and increased 5S RNA synthesis in cell-free extracts from polyoma-infected mouse kidney cell cultures.

In polyoma-infected mouse kidney cell cultures 5S RNA synthesis began to increase around 16 h, i.e. 7-9 h after the onset of polyoma T-antigen synthesis. The rate of polyoma-induced 5S RNA synthesis reached a maximum plateau around 25 h when it was 1.8-2.0 times higher than in mock-infected parallel cultures. Stimulation of 5S RNA synthesis in vivo thus coincided in time with the increase in total cellular RNA and protein. Cell-free extracts (S100) prepared at 15 h from mock-(S100-M) or polyoma-infected (S100-Py) mouse kidney cell cultures were indistinguishable with respect to protein concentration and 5S RNA synthesis, using a cloned somatic Xenopus borealis 5S gene as template. S100-Py extracted 25 h after infection contained 30% more protein and synthesized 1.5-2.0 times more 5S RNA than S100-M. Complete removal of the polyoma T-antigens from S100-Py by 3 cycles of immunoprecipitation with hamster anti-T serum remained without effect on stimulated 5S RNA synthesis. However, a linear relationship between 5S RNA synthesis and protein concentration of S100-M and S100-Py was observed.     

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

Adaptation to cycloheximide of macromolecular synthesis in Tetrahymena

Cycloheximide (CHI) at 10 ng/ml partially inhibited protein synthesis in exponential cultures of Tetrahymena Sp. At 20 ng/ml or greater, inhibition was complete. When protein synthesis was inhibited to any extent, cell division ceased immediately. In all instances where measured, synthesis of RNA and DNA also ceased. After a period of delay, cellular functions reinitiated in the order: (i) protein synthesis, (ii) DNA synthesis and, (iii) RNA synthesis and cell division. The delay in cell division was divided into three phases of: I, zero; II, low; and, III, fully recovered rates of exponential protein synthesis. The length of the three phases increased with increasing concentration of CHI Prior growth of cells for one generation in the presence of 7.5 ng/ml CHI (facilitation) eliminated phase I and slightly decreased phases II and III following subsequent challenge with an inhibitory concentration of CHI. Facilitation for six generations further decreased phases II and III. Protein synthesis and cell division were not inhibited during facilitation In the culture, succinate dehydrogenase activity did not increase during the delay but increased normally at the onset of division. In contrast, NADPH-cytochrome c reductase activity continued to increase for an hour after inhibition of protein synthesis, was constant for a period and did not increase again until an hour after reinitiatoin of cell division and RNA synthesis Inhibition of division of all cells was immediate and reinitiation of synthesis and cell division was non-synchronous.     

Protein synthesis in the early stages of cardiac hypertrophy

Cardiac hypertrophy, induced in rats by either tri-iodothyronine or isoproterenol, administered daily for 7 days, was monitored using several parameters. Both treatments increased RNA concentrations 24 hr after the first injection, while heart weight increased following 2 injections to 46% above control after 7 days. Cardiac protein synthetic activity, as determined by the rate of peptidyl-puromycin formation, was increased by both tri-iodothyronine and isoproterenol 24 hr after a single injection, implying an increase in the number of functional ribosomes. RNA activity (the rate of peptidyl-puromycin formation per unit RNA) remained constant, suggesting that neither accelerated rates of initiation or translation nor increased activation of pre-existing, non-translating ribosomes was involved in the observed increase in protein synthetic activity. In contrast, constant infusion of [14C] tyrosine indicated no change in protein synthetic rate 24 hr after a single tri-iodothyronine injection and decreased protein synthetic rate after isoproterenol injection. It is concluded that the use of [3H]puromycin to estimate protein synthetic activity may be a more sensitive procedure for detecting early changes in protein synthesis in cardiac hypertrophy than constant isotope infusion, owing to the problems associated with determining the precise precursor pool for protein synthesis in this latter method.     

The In Vitro Synthesis of RNA within the Rat Nodose Ganglion Following Vagotomy

Abstract: This report describes the application of an in vitro labelling procedure for the evaluation of changes in the uptake and incorporation of tritiated nucleotides into RNA of the rat nodose ganglion following crush injury of the cervical vagus nerve. Significant changes in the incorporation into 28S, 18S and 4S RNA were observed at 3 and 9 days after injury which confirms and extends our previous in vivo observations where [³²P]orthophosphate was used as the precursor. An early stimulation in the uptake of nucleotides, which was maximal at 2 days after injury, was also observed. Evidence is presented which indicates that this data reflects a real increase in RNA synthesis within the injured tissue concomitant with an increase in the uptake of nucleotide precursors which may reflect an increase in the nucleotide pool size. The transient nature of the rRNA synthetic responses and their occurrence prior to the peak of the chromatolytic changes suggest that there may be a shift in the distribution of ribosome types resulting in qualitative changes in protein production rather than an overall increase in protein synthesis resulting from an increased ribosome population.     

Two developmental stages of Neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for recovery.

At the heat shock temperature of 45 degrees C, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of Neurospora crassa. Both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. At the RNA level, however, these two developmental stages responded with different kinetics to elevated temperature. Heat shock RNAs (for hsp30 and hsp83) accumulated and declined more rapidly in spores than in mycelia, and during recovery spores accumulated mRNA that encoded a normal protein (the proteolipid subunit of the mitochondrial ATPase), whereas mycelia showed no increase in this normal RNA (for at least 120 min). Therefore, the resumption of normal protein synthesis in spores may depend upon accumulation of new mRNAs. In contrast, mycelial cells appeared to change their translational preference during continued incubation at elevated temperature, from a discrimination against normal mRNAs to a resumption of their translation into normal cellular proteins, exemplified by the ATPase proteolipid subunit whose synthesis was measured in the heat-shocked cells.     

Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by beta-ecdysone.

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.