Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

The $\text{in}$$\text{vivo}$ dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.     

Effects of selenium on RNA synthesis and content in rat pancreas.

In order to investigate the effect of selenium supplementation on RNA in the rat pancreas, the rate of in vitro incorporation of [3H]uridine into RNA by pancreas slices derived from two groups of rats fed either a low-selenium diet or a diet supplemented with 0.25 mg/kg selenium as selenite was examined. The RNA and lipid peroxide contents and glutathione peroxidase activity in homogenates from the pancreas were also determined. After feeding for 12-14 weeks, the rates of [3H]uridine incorporation were significantly higher in the pancreatic tissue from the selenium-supplemented diet group. Concomitantly, an increase in glutathione peroxidase activities and RNA content, and a reduction of lipid peroxides, were also found in the pancreatic tissue of the selenium-supplemented group. The results suggest that selenium supplementation at a level of 0.25 mg/kg selenium could promote RNA synthesis with an increase in glutathione peroxidase activity and a decrease of lipid peroxides.     

Enhancement of ribonucleic acid synthesis by chromium(III) in mouse liver

The effect of Cr(III) administration on hepatic RNA synthesis in mice was studied. It was found that Cr accumulated in mouse liver. Forty-eight hours after intraperitoneal injection of CrCl3 (0.005-5 mg Cr/kg body weight) approximately 10% of the administered dose per g of tissue remained. The accumulated Cr was still retained 64 days after administration (5 mg Cr/kg) with only a slight decrease. Approximately 20% of the hepatic Cr was detected in the nuclei. By administering CrCl3. RNA synthesis in mouse liver was markedly enhanced without altering the pool size of nucleotides. This enhancement was dose-dependent and statistically significant at doses of 0.05 (p less than 0.05), 0.5 (p less than 0.01), and 5 mg Cr/kg (p less than 0.01), and remained so for at least 16 days after administration of 5 mg Cr/kg. The synthesis of DNA and protein in mouse liver were not significantly changed by CrCl3 administration. On the other hand, Cr(VI) administration did not enhance but rather inhibited RNA synthesis in mouse liver. These results suggest that Cr(III) specifically enhances RNA synthesis in mouse liver.     

Effects of carbamylcholine and isoproterenol on electrolyte fluxes in perfused main duct of submandibular gland of reserpinized rat

Administration of reserpine (RES) at a dosage of 0.5 mg/kg body wt, ip daily for 7 days was found to lower the dose of carbamylcholine and isoproterenol that alters sodium and potassium transport by cells of the main duct of rat submandibular gland. In the perfused main excretory duct of the submandibular gland of the RES rat, administration of carbamylcholine at a dosage of 1 microgram/kg body wt, inhibited net efflux of sodium (17%) and administration of isoproterenol at a dosage of 2 micrograms/kg body wt increased net efflux of sodium (20%); these drugs, at the same dosages, did not induce significant change in electrolyte flux of normal rat. At a dosage of 5 micrograms/kg body wt, carbamylcholine decreased net influx of potassium (15%) in the RES rat but was without effect on normal rat. Isoproterenol at the dosage of 5 micrograms/kg body wt significantly inhibited net influx of potassium in both the RES rat and normal rat. The data suggested that the duct cells developed supersensitivity to sympathomimetic and parasympathomimetic stimulation after chronic RES treatment.     

Accumulation of activated lymphocytes in liver of endotoxin treated rabbits.

Intravenous administration of endotoxin in doses, 1.0 mg/kg, 0.1 mg/kg or 0.01 mg/kg caused activation (in the sense of nucleolar RNA synthesis) of the lymphocytes in peripheral blood, kidney and liver in rabbits. The evaluation of the ratio of lymphocyte count to the number of organ tissue cells leads to the conclusion that the accumulation of endotoxin activated lymphocytes occurs in liver.     

Secretion mode of the harderian gland of rats after stimulation by cholinergic secretagogues.

We studied the morphological changes in rat Harderian glands 30 min after injection of cholinergic secretagogues. In controls, the glands exhibited a tubuloalveolar structure with relatively wide lumina, in which some osmiophilic dense droplets exocytosed from glandular cells were observed. Also two types of glandular cells (type A cells and type B cells sometimes showing exocytotic figures of lipid-secretory vacuoles) and myoepithelial cells were recognized. After injection of carbamylcholine chloride (subcutaneously, 0.1 mg/kg body weight), which has both nicotinic and muscarinic actions, many of the alveolar lumina dilated and contained a small number of osmiophilic droplets. Exocytotic figures in both types of cells and a pronounced decrease in the number of vacuoles in the glandular cells were observed. However, there was no evidence of apocrine or holocrine secretion. The injection of the higher dose of carbamylcholine (1.0 mg/kg) caused fusion of secretory vacuoles in the apical cytoplasm and contraction of myoepithelial cells. Most alveoli showed no clear lumina; their centers were jammed with cytoplasmic fragments and accumulated secretory products. Massive discharge of cytoplasmic fragments containing some secretory vacuoles was often observed. This may be classified as apocrine secretion. Bethanechol chloride (subcutaneous injection, 1.0 mg/kg), a muscarinic agonist, stimulated the Harderian-gland secretion, and enhanced exocytosis was observed. The discharge from the glandular cells, following injection of various doses of carbamylcholine, were almost inhibited by atropine sulfate, a muscarinic antagonist. The present results suggested that the cholinergic systems regulate the secretion of rat Harderian-gland cells which have muscarinic receptors.     

Stimulation of hepatic protein synthesis in response to Mikania cordata root extract in carbon tetrachloride-induced hepatotoxicity in mice.

The effect of Mikania Cordata root extract was evaluated on the rate of hepatic protein synthesis in vivo in CCl4-induced liver damage. Pretreatment with the root extract (100 mg/kg, once daily for successive 5 days) showed a marked enhancement in the levels of hepatic DNA, RNA and protein content that were adversely affected with CCl4 treatment in the experimental mice. Increase in the total protein mass, fractional rate of protein synthesis (% of protein synthesized/day), total rate of protein synthesis (fractional rate x protein mass), ribosomal capacity (RNA/protein), ribosomal efficiency (rate/ribosome) and high turnover rate of protein (protein/DNA) in response to the pretreatment of the root extract in hepatic tissue indicated the tissue repair leading to a functional improvement of the hepatocytes that were disorganised with CCl4 intoxication.     

Dietary γ-aminobutyric acid affects the brain protein synthesis rate in young rats

Summary. The purpose of this study was to determine whether the γ-aminobutyric acid (GABA) affects the rate of brain protein synthesis in male rats. Two experiments were done on five or three groups of young rats (5 wk) given the diets containing 20% casein administrated 0 mg, 25 mg, 50 mg, 100 mg or 200 mg/100 g body weight GABA dissolved in saline by oral gavage for 1 day (d) (Experiment 1), and given the diets contained 0%, 0.25% or 0.5% GABA added to the 20% casein diet (Experiment 2) for 10 d. The plasma concentration of growth hormone (GH) was the highest in rats administrated 50 mg and 100 mg/100 g body weight GABA. The concentration of serum GABA increased significantly with the supplementation groups. The fractional (Ks) rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein + 0.25% GABA diet and still more 20% casein + 0.5% GABA compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA·d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. Our results suggest that the treatment of GABA to young male rats are likely to increase the concentrations of plasma GH and the rate of protein synthesis in the brain, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Reducing plasma HIV RNA improves muscle amino acid metabolism

We reported (Yarasheski KE, Zachwieja JJ, Gischler J, Crowley J, Horgan MM, and Powderly WG. Am J Physiol Endocrinol Metab 275: E577-E583, 1998) that AIDS muscle wasting was associated with an inappropriately low rate of muscle protein synthesis and an elevated glutamine rate of appearance (Ra Gln). We hypothesized that high plasma HIV RNA caused dysregulation of muscle amino acid metabolism. We determined whether a reduction in HIV RNA (> or =1 log) increased muscle protein synthesis rate and reduced R(a) Gln and muscle proteasome activity in 10 men and 1 woman (22-57 yr, 60-108 kg, 17-33 kg muscle) with advanced HIV (CD4 = 0-311 cells/microl; HIV RNA = 10-375 x 10(3) copies/ml). We utilized stable isotope tracer methodologies ([13C]Leu and [15N]Gln) to measure the fractional rate of mixed muscle protein synthesis and plasma Ra Gln in these subjects before and 4 mo after initiating their first or a salvage antiretroviral therapy regimen. After treatment, median CD4 increased (98 vs. 139 cells/microl, P = 0.009) and median HIV RNA was reduced (155,828 vs. 100 copies/ml, P = 0.003). Mixed muscle protein synthesis rate increased (0.062 +/- 0.005 vs. 0.078 +/- 0.006%/h, P = 0.01), Ra Gln decreased (387 +/- 33 vs. 323 +/- 15 micromol.kg fat-free mass(-1).h(-1), P = 0.04), and muscle proteasome chymotrypsin-like catalytic activity was reduced 14% (P = 0.03). Muscle mass was only modestly increased (1 kg, P = not significant). We estimated that, for each 10,000 copies/ml reduction in HIV RNA, approximately 3 g of additional muscle protein are synthesized per day. These findings suggest that reducing HIV RNA increases muscle protein synthesis and reduces muscle proteolysis, but muscle protein synthesis relative to whole body protein synthesis rate is not restored to normal, so muscle mass is not substantially increased.     

Hypertrophic response of the kidney in indomethacin treated rats.

Indomethacin, a potent inhibitor of prostaglandin (PG) synthesis administered to rats in a total dose of 2 x 4.5 mg/kg i.v. for 2 days enhanced compensatory renal hypertrophy following unilateral nephrectomy (UN). The same drug given to rats with intact kidneys in a total dose of 4 x 4.5 mg/kg i.p. in 2 days increased kidney weight renal RNA content and decreased renal acidic ribonuclease activity. It remains to be elucidated whether the suppression of PG synthesis and/or other pharmacological effects of indomethacin account for these findings.     

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.     

Effect of prednisolone on the intensity of nucleic acid synthesis by granulation tissue fibroblasts

An experimental study was made of the effect exerted by prednisolone in a dose of 4 mg/kg body weight on wound healing. It was shown that prednisolone reduces the period of inflammation, accelerates the formation of granulation tissue and epidermis. All these effects are accompanied by diminution of the mitotic activity of fibroblasts and dramatic increase in the intensity of RNA synthesis in them. Dislocation of newly synthetized RNA from the nucleus to the cytoplasm proceeds at a greater rate as compared to control. The differentiation of fibroblasts is accelerated.     

Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.

The rate of epidermal protein synthesis in vivo was determined in the hairless mouse by a method in which a large dose of [3H]phenylalanine (150 mumol/100 g body wt.) is administered via the tail vein. The epidermal free phenylalanine specific radioactivity rapidly rose to a plateau value which by 10 min approached that of plasma, after which it declined. This dose of phenylalanine did not of itself alter protein synthesis rates, since incorporation of co-injected tracer doses of [3H]lysine and [14C]threonine was unaffected. The fractional rate of protein synthesis obtained for epidermis was 61.6%/day, whereas values for liver and gastrocnemius muscle in the same group of mice were 44%/day and 4.8%/day respectively. When expressed on the basis of RNA content, the value for epidermis (18.6 mg of protein/day per mg of RNA) was approx. 3-fold higher than those for liver and gastrocnemius muscle. Topical administration of 0.1% triamcinolone acetonide increased the epidermal fractional protein synthesis rate by 33% after 1 day and by 69% after 7 days, compared with vehicle-treated controls. These effects were entirely accounted for by the increase in protein synthesis rates per mg of RNA. RNA/protein ratios were unaffected by this treatment.     

Qualitative and quantitative aspects of the biosynthesis of ribonucleic acid and of protein in the liver and the lung of the Syrian golden hamster

1. The incorporation of orotic acid and of uridine into total RNA was measured in vivo in liver and lung of the Syrian golden hamster. Specific activities of total acid-soluble UMP were measured in both organs. An estimation of the rate of RNA biosynthesis showed that hamster lung synthesizes RNA at about one-half of the rate of that of hamster liver. 2. The apparent K(m) and V(max.) values of a few enzymes involved in pyrimidine biosynthesis were measured in the 100000g supernatants of liver and lung. The apparent K(m) values were very similar in both organs. From the estimated V(max.,) it was concluded that hamster lung cells have less capacity to metabolize orotic acid than have liver cells. 3. A time-response and a dose-response study showed that actinomycin D inhibits pulmonary RNA synthesis as efficiently as hepatic RNA synthesis. 4. Protein synthesis, measured as the incorporation of leucine, was inhibited in both organs 30min after a dose of 2mg of cycloheximide/kg. The dose-response patterns were similar in both liver and lung 3h after cycloheximide. 5. It is concluded that RNA and protein synthesis in vivo in hamster lung are very similar to the corresponding reactions in liver. Alterations of RNA and protein synthesis by toxic agents can therefore be evaluated in lung with a similar approach to that used to study the pathological biochemistry of liver.     

CORRELATION BETWEEN ELECTRICAL ACTIVITY AND SPLITTING OF PHOSPHOLIPIDS BY SNAKE VENOM IN THE SINGLE ELECTROPLAX

Abstract— Cottonmouth moccasin snake venom (SV) was applied to the innervated membrane of the isolated single cell of the Sachs electric organ (electroplax) of the electric eel, Electrophorus electricus. Concentrations as low as 0.05 μg/ml irreversibly antagonized depolarization by carbamylcholine, whereas concentrations of 0.1 mg/ml or higher were required to directly and irreversibly depolarize and block electrical excitation. The active component of the venom was stable to boiling at acid pH, destroyed by boiling at alkaline pH and nondialyzable and corresponded to those fractions containing maximum phospholipase A activity demonstrable when isolated by paper electrophoresis and Sephadex filtration. Phospholipase C and lysolecithin in concentrations of 1 mg/ml and 0.2 mg/ml, respectively, depolarized and blocked electrical excitation, whereas lower concentrations did not antagonize depolarization by carbamylcholine. Triton X-100 (0.01 mg/ml) antagonized carbamylcholine, whereas 10-fold higher concentrations directly blocked electrical excitation. Hyaluronidase had no effect on resting or action potential but decreased the depolarizing response to carbamylcholine. At minimal concentrations which blocked the depolarizing response to carbamylcholine, SV caused only slight splitting of phospholipids in single cells of the Sachs organ. A concentration (1 mg/ml) of SV which blocked electrical excitation caused 80–100 per cent splitting of lecithin, phosphatidylethanolamine and phosphatidylserine, the three principal phospholipids of the electric tissue. Similar percentages of splitting of the latter two phospholipids but only about one-third of the lecithin occurred at SV concentration of 0.1 mg/ml. These results indicate that electrical excitability in the eel electroplax can be maintained in the presence of extensive phospholipid splitting. Depolarization and block of electrical excitation by relatively high concentrations of SV may have resulted from splitting of phospholipids, especially lecithin, or may have reflected action of lysophosphatide detergents produced as a result of the action of phospholipase A upon membranal phospholipids.     

Effect of amantadine on the rate of dopamine synthesis in rat corpus striatum.

Abstract— The effect of amantadine on the rate of dopamine synthesis in rat corpus striatum was determined by three methods. (1) Measuring the rate of decline of endogenous dopamine following inhibition of synthesis with a-methyltyrosine (α-MT); (2) Measuring the rate of conversion of [3,5-³H]tyrosine to ³H-labelled catechols under conditions of an initial rate; and (3) measuring the levels of homovanillic acid (HVA), the principal metabolite of brain dopamine. Endogenous dopamine levels were 68-1 n-mole/g with a control synthesis rate of about 21 n-mole/g/h as determined using either α-MT or [3,5-³H]tyrosine. Amantadine had no effect on synthesis at doses up to 100 mg/kg using α-MT and [3,5-³H]tyrosine. HVA levels were unaffected after 30 mg/kg drug, but were elevated 48%(P < 005) after 100 mg/kg of drug. By contrast apomorphine reduced and haloperidol increased synthesis as determined by all three methods. It is concluded that amantadine has no marked effect on dopamine synthesis in rat corpus striatum.     

Effect of Cu2+ and Ni2+ on the intensity of RNA synthesis in corn seedlings]

It has been found that Cu and Ni at contents of 17 mg/kg and 64 mg/kg dry weight respectively in leaves tissues have no visible effect on morphological and physiological characteristics of plants and increase [3H]uridine incorporation in various types of RNA. Under these conditions synthesise of nuclear RNA has been found 1.8-2.4 more intense, transport RNA -2.0-2.8 intense and ribosomal RNA-1.2-2.8 intense in comparison with control. Analysing our data it is possible to suggest that the activation of the RNA synthesis under the heavy metals influence may be one of the protective mechanism connected with the plant cell adaptation to the stressful conditions.     

Effects of adding dietary lysine to a low gluten diet on the brain protein synthesis rate in aged rats

The purpose of this study was to find whether the addition of dietary lysine affected the rate of brain protein synthesis in aged rats fed on a gluten diet. Experiments were done on two groups of aged rats (30 wk) given the diets containing 5% gluten or 5% gluten + 0.3% lysine for 10 d. The fractional rates of protein synthesis in brain, liver, and kidney increased with an addition of dietary lysine. In brain, liver, and kidney, the RNA activity [g protein synthesized/(g RNA x d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the addition of the limiting amino acid for the low quality protein elevates the rate of protein synthesis in the brain of aged rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.