Specific Activity Of RNA Research Articles

Growth and protein turnover in Atlantic salmon ( Salmo salar L.); the effect of dietary protein level and protein particle size

In modern fish feeds, the protein sources consist of denaturated finely ground ingredients. From the literature, it has been reported that use of coarsely chopped, but not denaturated, fish as the dietary protein source gave better growth performance and protein utilisation. Growth, feed utilisation and protein turnover using two different fish meal particle sizes (micro- or coarse-grounded) at three dietary protein concentrations (30%, 35%, and 45%) were studied in individually tagged Atlantic salmon in a 3-month growth experiment. At the end of the experimental period, 14C- l-lysine was injected intraperitonally and dorsal muscle samples were taken at 2- and 4-h post-injection. Incorporation of 14C- l-lysine into muscle protein, RNA, DNA and water soluble protein was analysed from samples of muscle tissue. Only small effects on growth rate, feed conversion rate, protein and energy retention, and nitrogen and fat digestion were found. During the growth experiment, large individual variations in growth rates were observed, which did not correlate to the initial body weight. The total RNA content expressed as RNA amount per unit of DNA (RNA:DNA ratio) did not reflect the specific RNA activity, and individual growth rate was not correlated to the specific RNA activity or RNA:DNA ratio and only poorly to the relative incorporation rate of amino acids. Growth rate was, however, correlated to the relative efficiency of protein synthesis. The results indicate that the protein catabolism is more important for net protein deposition and growth than protein anabolism.

Blood-brain barrier carrier-mediated transport and brain metabolism of amino acids.

The transport of neutral amino acids through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, is an important control point for the overall regulation of cerebral metabolism, including protein synthesis and neurotransmitter production. The Michaelis-Menten kinetics of BBB amino acid transport have been investigated in vivo with the brain uptake index (BUI) technique, and in vitro with the isolated human brain capillary preparation. The only amino acid that is albumin-bound is tryptophan, and the majority of albumin-bound tryptophan in the plasma is available for transport through the BBB via an enhanced dissociation mechanism that operates at the surface of the brain capillary endothelium. The availability in brain of amino acids is predicted from the BBB Km values to be sharply influenced by supra-physiological concentrations of phenyalanine in the 200-500 microM range. Moreover, the measurement of cerebral protein synthesis with an internal carotid artery perfusion technique and HPLC-based measurements of aminoacyl-transfer RNA specific activities shows an inverse relationship between cerebral protein synthesis and plasma phenyalanine concentrations in the 200-500 microM range. These findings indicate the neurotoxicity of hyperphenylalninemia is not restricted to the phenylketonuria range of approximately 2000 microM, but is exerted in the supra-physiological range of 200-500 microM.

The effects of salinity, hypophysectomy and hormone administration on gill RNA metabolism of the euryhaline teleost, Fundulus kansae

1. 1. The specific activity ( 32P incorporation) of the gill ribosomal RNA and soluble RNA fractions was decreased in both one third and full strength sea-water adapted animals compared to those held in fresh water. 2. 2. Hypophysectomy of sea-water adapted fish increased the specific activity of the soluble tissue nucleotide fraction; hypophysectomy may have altered the ratio of incorporation of 3H-uridine and 14C cytosine into r-RNA. 3. 3. ACTH lowered the specific activity of the soluble tissue nueleotide pool in hypophysectomized, sea-water adapted fish; ACTH altered the U C ratio of the r-RNA. 4. 4. Prolactin injections caused little change in the U C ratio in hypophysectomized, sea-water adapted fish, but probably did cause a decrease in RNA specific activity. 5. 5. It is suggested that the lack of conclusive influences of ACTH and prolactin on gill RNA metabolism indicates that exogenous factors, such as ion balances and ratios, may play a key role in RNA changes associated with osmoregulation.

Alterations of pyrimidine and nucleic acid synthesis during adaptive growth of Liver induced by nafenopin, a peroxisome proliferator. An in vivo study

The de novo synthesis of pyrimidine nucleotides in the rat liver after administration of nafenopin (NFP) was studied with the aid of [14C]orotic acid; the utilization of preformed nucleosides (salvage pathways) was followed using the [14C]cytidine and [14C]thymidine. A single dose (400 mg/kg) as well as repeated doses (100 mg/kg/day) of NFP increased the concentration of the cytidine and uridine components of the acid-soluble extract (ASE) of rat liver. Increase in the concentration of the cytidine components preceded the increase in the uridine components. The uptake of [14C]cytidine by the liver of rats that had been given a single dose of NFP was observed 24 h after the administration of the drug and a decrease followed after this period. The specific activity of RNA and DNA cytosine paralleled the changes of the specific activity of ASE. A single dose of NFP had no marked effect on the uptake of [14C]orotic acid. The specific activity of the uridine components of ASE remained unaltered for 2 days. After this period it decreased because of an increase in the amount of the soluble uridine components. A mild drop of the specific activity of cytidine components of ASE occurred on the second day, the total radioactivity of cytidine components increased 24 h after the administration of NFP. The specific activity of DNA pyrimidines was markedly increased 24 h after administration of the drug. On the fourth day the specific activity of DNA cytosine in the experimental group was the same as in the control group, whereas the activity of DNA thymine was lower. Following repeated administration of NFP (100 mg/kg/day) a decreased uptake of [14C]orotic acid was observed; its utilization for the synthesis of the uridine components of ASE, expressed as total radioactivity of soluble uridine components, was continuously suppressed. No changes in the specific activity of cytidine components were observed. The specific activity of DNA cytosine and thymine was distributed unevenly. During later periods of the drug action the specific activity of cytosine increased whereas the activity of thymine was lower. This phenomenon may be accounted for by the formation of a thymidylate precursor, dUMP, directly from uridine phosphates.

Direct determination of the specific activity of RNA uniformly labeled with 32P

A simple method for the direct determination of the specific activity of RNA uniformly labeled with 32P is described. The procedure is based on the premise that upon disintegration of 32P to 32S, the phosphodiester bond is broken. Analysis of the rate of decay of the full-length molecule by gel electrophoresis and autoradiography can accurately determine the “intramolecular specific activity” of the RNA. An equation that predicts the relative intensity of the intact RNA molecules remaining as a function of time is presented. These predictions are confirmed using in vitro-synthesized RNA labeled at a known specific activity. This procedure has been used to determine the intramolecular specific activity of RNA labeled in vivo in yeast. It can also be employed to choose the best conditions for experiments utilizing uniformly labeled RNA or single-stranded DNA and requiring the detection of intact molecules.

Bleomycin-induced pulmonary fibrosis in hamsters. An alveolar macrophage product increases fibroblast prostaglandin E2 and cyclic adenosine monophosphate and suppresses fibroblast proliferation and collagen production.

Bleomycin-induced pulmonary fibrosis in hamsters is associated with collagen accumulation that results from increased lung collagen synthesis rates. However, 1-2 wk after intratracheal instillation of bleomycin, lung collagen synthesis rates decline toward control values. To evaluate the potential role of the bronchoalveolar macrophage in regulating lung collagen production, we studied the effects of macrophages from normal and bleomycin-treated hamsters upon fibroblasts in vitro. We observed: (a) Medium from macrophage cultures decreased fibroblast [3H]thymidine incorporation and nondialyzable [3H]hydroxyproline production in a dose-dependent manner. Fibroblast cell counts were decreased in exposed cultures, and fibroblast viability was unchanged. Procollagen prolyl hydroxylation and prolyl-transfer RNA-specific activity were not altered by macrophage medium; this indicates that [3H]hydroxyproline reflects collagen production rate under the experimental conditions. (b) The suppressive effect of macrophage medium was selective for collagen since collagen production decreased more than noncollagen protein production. (c) Medium from bleomycin-treated hamster macrophages suppressed fibroblast proliferation and collagen production to a greater degree than medium from normal hamster macrophages. (d) Fibroblast suppression by macrophage medium was associated with increased fibroblast endogenous prostaglandin E2 production and intracellular cyclic AMP (cAMP). (e) Incubation of fibroblasts with indomethacin before exposure completely inhibited prostaglandin E2 production and increases in cAMP, and eliminated suppression of fibroblast proliferation and collagen production. The macrophage-derived suppressive factor has an apparent molecular weight of 20,000-30,000 and is heat stable. It does not appear to be species restricted since both hamster and human lung fibroblasts are similarly suppressed. It is at least in part preformed in macrophages obtained by lavage, but its production can also be stimulated in vitro. We concluded that alveolar macrophages release a product that stimulates endogenous fibroblast prostaglandin E2 production and cAMP formation with resultant suppression of fibroblast proliferation and collagen production. Enhanced release of suppressive factor by macrophages during a time when lung collagen production is declining in bleomycin-induced pulmonary fibrosis suggests that macrophages may limit collagen accumulation in pulmonary fibrosis.

RNA, Proteins and Polyamines During Tube Growth in Germinating Apple Pollen

Variations of RNA, protein, and free- and trichloroacetic acid-soluble bound polyamine levels were determined during tube growth in germinating Malus domestica Borkh. cv. Starkrimson pollen.During rehydration of pollen no marked differences were observed, whereas, during germination, RNA, proteins, and polyamines showed parallel decreases. At the same time, there was synthesis of RNA and polyamines as indicated by use of labeled precursors. The data indicate that during germination: (a) the genes for rRNA, tRNA, and probably mRNA are active; (b) the enzymes involved in polyamine biosynthesis are very active. High levels of free arginine during the first 15 minutes were observed, probably in response to a demand for this precursor in polyamine biosynthesis. Moreover, profiles of the variations in the specific activities of RNA and polyamines showed similar patterns. The results indicate that biosynthesis of RNA and polyamines precedes tube emergence. The possible role of these compounds, which are known to be released into the medium in the progamic phase of the fertilization processes, is considered.

Genetic modulation of RNA metabolism in Drosophila. III. Requirement for an rDNA-deficient X chromosome in YbbSuVar-3-mediated increases in RNA synthesis.

Males of the genotype car bb/YbbSuVar-5 have an approximately two-fold increase in the rate of accumulation of 4S, 5S, 18S plus 28S and poly-A+ RNA molecules when compared to males of the genotype car bb/Ybb- (CLARK, STRAUSBAUGH AND KIEFER 1977; CLARK and KIEFER 1977). Experiments were designed to determine if the modulation of RNA metabolism by YbbSuVar-5 requires an rDNA deficiency on the X chromosome. Synthetic rates for 4S, 5S, and 18S plus 28S RNA's were measured in the genotypes Sam + iso/Ybb- and Sam + iso/YbbSuVar-5 by injecting adult males with 3H-uridine, allowing them to incorporate label for two hours, fractionating extracted RNA by polyacrylamide gel electrophoresis and determining the specific activities of RNA's (dpm/microgram RNA). The results of these determination showed that the synthetic rates for 4S, 5S, and 18S plus 28S RNA's are the same in these two genotypes, demonstrating that the YbbSuVar-5 chromosome does not increase transcription when paired with an X chromosome that is wild type for rDNA. Saturation hybridization was used to measure the rDNA content in several genotypes. These results showed that Sam + iso/YbbSuVar-5 males and Sam + iso/Ybb- males have equivalent amounts of rDNA.--These results are consistent with the suggestion that the Ybb- and YbbSuVar-5 chromosomes have similar amounts of rDNA. The nature of compensatory strategies in rDNA-deficient genotypes is discussed.

Maternal dietary deficiency and its effect on the metabolism of nucleic acids and proteins. "Effect of exchanging the young, during the lactation period, between the control and undernourished female rats".

The effect of maternal dietary deficiency on the metabolism of nucleic acids and proteins was studied by exchanging the pups of control and undernourished dams during the lactation period. In the pups of control dams fostered by undernourished dams during the lactation period (E3), it was observed that the body and organ weight, and RNA, DNA, and protein content failed to increase normally. Contrary to this, the free leucine and nucleotide contents were higher and their specific activities lower in the plasma and various organs of the E3 group as compared with the control group.Specific activity of protein was higher in the liver, brain, kidney, and lung, and was lower in the spleen and heart of the E3 group as compared with the control group. Specific activity of RNA was higher in the liver, spleen, and lung, and was lower in the brain, kidney, and heart of the E3 group as compared with the control group.In the pups of undernourished dams fostered by the control dams during the lactation period (E1), the body and organ weights, the RNA, DNA, and protein content, the content of free leucine and nucleotides as well as their specific activities, and the specific activity of protein and RNA were partially or completely restored. However, the DNA content of the brain remained unchanged in comparison with those pups of undernourished dams nursed by their own mother (E2). In the brain, kidney, spleen, and lung of the E1 group, the specific activity of RNA increased considerably and even exceeded the control values.The radioactivity results discussed above clearly demonstrate an accelerated metabolism of protein and RNA in the various organs of the E3 group and a partial or complete normalization in the E1 group.

Studies on Ribosomal RNA Synthesis in Vivo in Rat Liver during Short-Term Protein Malnutrition

To better understand the increased incorporation of [14C] orotate into rRNA, as detailed study of liver rRNA synthesis was performed on young rats fed a 6% casein diet for 1 week. From 10 to 180 minutes after i.v. injection of [14C] orotate, nucleolar RNA specific activity of malnourished rats was 1.6 times higher than controls. Polyacrylamide gel electrophoresis revealed that 45S pre-rRNA from malnourished rats was synthesized more rapidly than controls. Specific activities of other nucleolar RNA species was increased but the time of appearance of labeled peaks did not change. The cellular cytoplasmic rRNA pool was reduced to 70% of controls, the time of appearance of labeled rRNA in the cytoplasm was the same, but cytoplasmic rRNA specific activity was 2.1 times greater than controls. Absorbance profiles and radioactivity patterns of nucleoplasmic RNA were similar in the two groups of rats. Nucleoplasmic radioactivity patterns were complex and heterodispersed, and the amount of radioactivity was greatest in RNA from malnourished rat liver. Our data indicate that short-term dietary protein restriction leads to cellular events which increase the synthesis of nuclear RNA. Nucleolar RNA is processed and transported to the cytoplasm in a manner similar to controls.

Effect of disseminated necrosis of the heart on uptake of radioactive precursors into RNA of internal mitochondrial membranes of the rat myocardium

The role of the internal mitochondrial membranes of the rat myocardium as the site of uptake of radioactive precursors into mitochondrial RNA was studied. The kinetics of the changes in specific RNA activity of the internal mitochondrial membranes of the myocardium was found to differ in vivo and in vitro. Necrosis of the myocardium, in experiments in vivo and in vitro, caused significant changes in the specific RNA activity of the mitochondrial membranes in the same direction.

Activation of ribosomal and messenger RNA synthesis in excised Jerusalem artichoke tuber slices

RNA synthesis was studied in Jerusalem artichoke (Helianthus tuberosus L.) tuber slices immediately following excision and during the early period of aging in water. Incorporation of [(3)H]adenosine into RNA was detected as early as 20 min after excision. Measurement of the specific activities of RNA (cpm/μg) and of ATP showed that RNA synthesis proceeded at a constant rate for the first several hours of aging and then increased moderately. [(3)H]adenosine was incorporated into polysomes throughout the aging period examined. Sucrose gradient fractionation of EDTA-dissociated polysomes showed that during the first 2 h of aging most of this incorporation was not into ribosome subunits but into presumed mRNA. Autoradiographic analysis of [(3)H]adenosine labelled nuclei showed that this was caused, at least in part, by a delay in the onset of rRNA synthesis synthesized during this time chromatographed as poly(A)-RNA on oligo(dT)-cellulose, indicating that a large part of the mRNA was not polyadenylated.

Carbaryl effects on growth and development in suspension cultures of wild carrot.

Carbaryl has been demonstrated to inhibit growth of carrot suspension cultures at 10 and 20 ppm. The specific activities of RNA and protein increased at 20 ppm. Total RNA was relatively unchanged at any concentration while total protein was reduced considerably at 20 ppm. Embryo development was inhibited by increasing concentrations of carbaryl. Thin layer chromatography indicated considerable degradation of carbaryl to 1-naphthol. When 1-naphthol was tested in this system it was shown to elicit the same inhibitory effects on growth as carbaryl, suggesting that the 1-naphthol residue is itself a potent inhibitor of tissue growth and development.

Synthesis of RNA and protein, with ribonuclease activity, in carrot roots infested with Meloidogyne hapla chitw

Roots of 5-month-old healthy and Meloidogyne hapla infested carrot cv. Slendero (tolerant to this nematode) and cv. Perfekcja (sensitive to this nematode) were analysed. In galls which proliferated from rhizogenic plaques of the storage roots of both cultivars (1) RNA and protein contents were higher than in other tissues, (2) the rate of 14C-uracil incorporation into RNA on a fresh weight basis was also higher than in other tissues and (3) the specific radioactivity of protein was lower than in all other tissues. When galled side roots of cv. Slendero were compared with healthy roots (1) RNA did not accumulate in spite of a doubling of RNA synthesis at the time of analysis, (2) the protein content was about 70% higher and specific radioactivity of protein lower by 60% and (3) RNase activity per mg protein decreased by 80%. When galled side roots of cv. Perfekcja were compared with healthy roots: (1) RNA and protein contents were increased 20 and 30%, respectively, (2) RNA synthesis was stimulated, whereas specific radioactivity of protein was not modified though increased as ct/min/mg fr. wt and (3) the specific activity of RNase was halved. In secondary phloem and secondary xylem tissues of the storage root of infested carrot cv. Slendero RNA synthesis was inhibited, protein synthesis stimulated and RNase activity decreased in comparison with healthy plants. In corresponding tissues of infested cv. Perfekcja the specific activity of RNA and protein was not affected, and the specific activity of RNase increased. Thus infestation, resulting in gall formation, also results in the accumulation of RNA in the infested tissue. This RNA accumulation occurs because of stimulated RNA synthesis, decreased RNA breakdown and protein accumulation. Infestation has variable effects on the storage tissues.

Action of Cyproterone Acetate on the Accessory Organs of Reproduction in Prepubertal and Sexually Mature Rats

The effects of cyproterone acetate on the accessory organs of immature, prepubertal, and sexually mature adult rats were studied. Administration of CA to immature 30-day-old rats for 15 days markedly decreased the silaic acid in the caput and cauda epididymides and decreased the secretory activity of the accessory glands. Treatment of adult rats with CA for 15 days caused an increased incorporation of 3H-uridine into RNA and of 3H-phenylalanine into protein in the caput and cauda epididymides. The percentage of increase in the specific activity of RNA and protein was higher in the cauda epididymidis than in the caput epididymidis.

Elevated serum growth hormone in protein-deprived rats, and decreased liver RNA after hypophysectomy.

Serum growth hormone was significantly higher in rats fed on a non-protein than on a 25% casein diet. In both dietary groups, hypophysectomy provoked a decrease in the RNA/DNA ratio of the liver, and, especially in the protein-deprived rats, a decrease in the RNA synthesis per liver cell, measured as the product of the RNA/DNA ratio, and the ratio of specific RNA activity to specific nucleotide activity, two hours after administration of [3H]orotic acid. In the protein-deprived rats, full reconstitution of these figures was obtained with a combination of growth hormone, hydrocortisone and triiodothyronine, but not quite with growth hormone alone. The possible role of growth hormone in protein deprivation is discussed.

RNA metabolism in the guinea pig infected with the nematode Trichostrongylus colubriformis

The concentration of RNA/mg DNA fell progressively in skeletal muscle, but was unchanged in the liver during infection of the guinea pig by the nematode Trichostrongylus colubriformis. The rate of RNA turnover was increased in muscle and liver. The specific activity of RNA 24 hr after the injection of 3H-uridine was unchanged in muscle, but increased in liver. While the concentrations of RNA precursors were unchanged in liver. The activity of muscle RNA polymerase was decreased, but that of liver was unchanged by the infection. From these results, it was concluded that the fall of the concentration of muscle RNA was due to both the increased rate of turnover and a decreased rate of synthesis, whereas the increased rate of turnover of liver RNA was approximately equal to the increased rate of synthesis. The significance of these findings is discussed in relation to changes of muscle and liver protein metabolism in this nematode and in bacterial or viral infections, as well as those that occur following reduced feed intake.

Polyamine and nucleic acid metabolism in myocardial hypertrophy of the overloaded heart

The metabolism of polyamines and nucleic acids in the hearts of rats undergoing physical activity for 2 hours once daily for 5 days has been studied. The results show a significant rise of the specific radioactivity of the polyamines (spermine and spermidine); this phenomenon reaches its peak on the second day of muscular activity. Under the same experimental conditions, the enzymic activity of amine-oxidase was evaluated, and increased on the second day of exercise; after reaching the maximum level, it remained constant until the end of the experiment. The specific activity of RNA of all the sub-cellular fractions of the heart showed a progressive increase with the duration of muscular activity. Experiments carried out with the perfused heart showed that spermine, at the concentration of 0.3 m m, stimulated the incorporation of [ 3H]ribose into RNA. The results suggest that the polyamines may be involved in the mechanism of myocardial RNA synthesis.

RNA metabolism during metamorphosis of Drosophila hydei

Determinations of total protein and RNA at various times after the beginning of the pharate pupal stage of Drosophila hydei, revealed an increase in both substances during the first 25 hr and a sudden decrease thereafter until 52 hr. From this time on the total amount of both protein and RNA increases slightly until emergence of the flies at 160 hr after the beginning of the pharate pupal stage. A similar pattern of changes was recorded for the total radioactivity as well as the specific activity of RNA labelled with 3H-uridine after the injection of the isotope immediately before the beginning of the pharate pupal stage. The migration profile of RNA labelled with 3H-uridine during larval development, revealed that shortly after the onset of the pharate pupal stage an essentially normal larval pattern consisting of major fractions of 28, 18, 8 to 9, and 4 to 7 S RNA. At 52 hr only the low molecular weight RNA was present. The ‘normal’ pattern was restored at the time of emergence of the flies, indicating re-utilization of degradation products of previously labelled RNA.

Metabolism of RNA and pyrimidine nucleotides in the uterus during the early decidual cell reaction.

SUMMARY Changes in the metabolism of pyrimidine 5′-nucleotides and RNA in the mouse uterus during the oil-induced decidual cell reaction were investigated. During the first 20 h after the intraluminal injection of 10 μl sesame oil the amounts of HClO4-soluble nucleotides and uridine 5′-nucleotides in the treated horn increased by 55% and 115%, respectively. During the same interval RNA increased by 101%, while the increase in protein was much smaller, and the amount of DNA remained almost unchanged. Doses of [2-14C]uridine, [5-3H]cytidine and [5-3H]orotic acid were administered to mice 20 min before killing. After [2-14C]uridine injection the specific activity of the uridine 5′-nucleotide fraction 20 h after the oil stimulus had not changed, while the specific activity of RNA increased by 43% during the 20-h interval. Similar results were obtained when [5-3H]-cytidine was injected. In contrast, after an injection of [5-3H]orotic acid the specific activities of both the uridine 5′-nucleotide and RNA fractions decreased with increasing time after the intraluminal injection of oil. When approximate rates of RNA synthesis were calculated independently from the data for [2-14C]uridine and [5-3H]orotic acid incorporation in these two fractions, the results agreed well, and indicated that the rate of synthesis of RNA in the decidualizing uterus increased by 64% and 163% at 8 and 20 h respectively after the intraluminal administration of sesame oil.