Changes In RNA Content Research Articles

Relationship of Protein Synthesis to mRNA Levels in the Liver of Chicks under Various Nutritional Conditions

The rate of liver protein synthesis in vivo and hepatic messenger ribonucleic acid (mRNA) levels were measured to examine the mechanisms regulating liver protein synthesis in chicks under various nutritional conditions. Fractional synthesis rate (FSR) of liver protein was measured by using a large dose injection of L-[4-3H]phenylalanine. Poly(A)+RNA was extracted as mRNA from total RNA using poly(U)-Sepharose 4B affinity chromatography. The influence of varying dietary protein levels (0–60%) on protein synthesis and mRNA concentration in the liver of chicks was examined. Both FSR (%/d) and absolute synthesis rate (ASR, mg/d) of liver protein increased with increasing dietary protein levels from 0 to 20% (the dietary protein requirement), whereas when dietary protein increased from 20 to 40%, both rates decreased significantly. This change was accounted for by changes in RNA concentration (RNA:protein ratio) and hepatic mRNA content. Second, the time course of changes in liver protein synthesis and hepatic mRNA content were measured in force-fed chicks that had been deprived of food for 2 d. Liver protein synthesis (FSR) was significantly greater 30 min after refeeding compared with that of unfed chicks. Liver protein synthesis (FSR) correlated with the activity of RNA to synthesize protein (r = 0.61, P < 0.001), and ASR was correlated with hepatic mRNA content (r = 0.37, P = 0.030). Third, experimental diets containing individual nutrients were force-fed to chicks after 2 d of food deprivation. When chicks were refed carbohydrate and protein diets, liver protein synthesis (FSR) tended to be greater by 24% (P = 0.063) and 22% (P = 0.075) at 30 min after refeeding compared with that of unfed chicks, respectively. These findings indicate that changes in the rate of liver protein synthesis in vivo due to the alteration in nutritional conditions are, at least partially, accounted for by not only RNA concentration and the activity of RNA synthesizing protein but also hepatic mRNA levels.

Protein synthesis, growth and energetics in larval herring (Clupea harengus) at different feeding regimes

Rates of growth, protein synthesis and oxygen consumption were measured in herring larvae, Clupea harengus, in order to estimate the contribution that protein synthesis makes to oxygen consumption during rapid growth at 8°C. Protein synthesis rates were determined in larvae 9 to 17 d after hatching. Larvae were bathed in (3)H phenylalanine for several hours and the free pool and protein-bound phenylalanine specific radioactivities were determined.Fractional rates of protein synthesis increased 5 to 11 fold with feeding after a period of fasting. Efficiencies of retention of synthesized protein were approximately 50% during rapid growth. Rapid growth in herring larvae thus appears to be characterized by moderate levels of protein turnover similar to those obtained for larger fish. Increases in growth rate occurred without changes in RNA concentration, i.e., the larvae increased the efficiency of RNA rapidly. Oxygen consumption rates were not correlated with growth rates. Protein synthesis was estimated to account for 79% of the oxygen consumption, and energy costs of protein synthesis were high, i.e., about 98 mmole O2 g(-1) protein synthesized.

Growth rates, nucleic acid concentrations, and [formula omitted] ratios of juvenile white shrimp, Penaeus vannamei boone, fed different algal diets

Macro- and microalgae are potentially important food resources for penaeid shrimp inhabiting coastal nursery grounds and aquaculture ponds. However, there are few data on the nutritional contribution of different algal species to juvenile shrimp growth. In this study, changes in whole body weight, RNA and DNA concentrations, and RNA DNA ratios of juvenile white shrimp, Penaeus vannamei Boone, fed different algal species were compared to assess the nutritional contribution of each species to shrimp growth. Shrimp fed a diatom culture composed primarily of Chaetoceros sp. were significantly heavier ( p < 0.05) than shrimp fed a monoculture of the green alga, Nannochloropsis oculata, fronds from the leafy macroalga, Ulva sp., or fronds from the filamentous macroalga, Enteromorpha sp. after 5 days. In addition, RNA/DNA ratios were significantly greater ( p 0 ̌ .05 ) in abdominal muscle tissue from diatom-fed shrimp. About 76% of the variation in shrimp growth rate could be explained by changes in RNA concentration, whereas RNA DNA ratio accounted for about 80% of the variation in growth rate. Diatoms can contribute substantially to short-term shrimp growth, and are probably important in coastal nursery grounds and aquaculture ponds when other food resources are scarce or to supplement available food.

Regulation of rDNA transcription by insulin in primary cultures of rat hepatocytes.

The studies presented herein were designed to investigate the role of insulin in maintaining the steady-state number of ribosomes in primary cultures of rat hepatocytes. The RNA content of hepatocytes maintained in the presence of insulin did not change during the 7 days of culture. In contrast, the RNA content declined significantly following 2 days of insulin deprivation and after 4 days without insulin reached a new steady-state value that was approximately 60% of that observed for hepatocytes maintained in the presence of the hormone. Following addition of insulin, the RNA content of insulin-deprived hepatocytes started to increase within 6 h and was restored to the control value within 48 h. The amounts of 18 and 28 S RNA, and thus the number of ribosomes, changed in concert with the total RNA content. Furthermore, synthesis of total cellular protein responded in parallel to the changes in RNA content, suggesting that the number of ribosomes was the primary determinant of protein synthesis under the conditions of these experiments. About one-half of the increase in RNA content following the addition of insulin to insulin-deprived cells could be accounted for by a reduction in the rate of degradation of ribosomes. The remainder of the change in RNA content must have resulted from an increase in ribosome biogenesis. This possibility was confirmed by measuring [3H]uridine incorporation into ribosomal RNA present in mature ribosomes. The rate of synthesis of ribosomal RNA was reduced in parallel with the fall in RNA content in insulin-deprived hepatocytes and was restored to the control value within 3 h of the addition of insulin. Alterations in the synthesis of ribosomal RNA in response to insulin deprivation and replacement were associated with parallel changes in transcription of rDNA as measured in nuclear run-on assays using a DNA probe corresponding to the external transcribed spacer region of rat rDNA. The data demonstrate that insulin regulates the number of ribosomes in primary cultures of rat hepatocytes by accelerating the rate of transcription of rDNA and by slowing the rate of ribosome degradation.

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 exercise on tissue protein synthesis in rats.

The effect of exercise on the protein metabolism in skeletal muscles (gastrocnemius and soleus), liver and small intestine was investigated in rats. Treadmill treatment for 7 d resulted in atrophy of the liver and small intestine, which was associated with a reduction in protein content. The rates of protein synthesis in the liver and small intestine were significantly suppressed in rats subjected to exercise. The change in protein synthesis in the visceral organs was mediated by the change in RNA activity (protein synthesis per unit RNA) but not by the change in RNA concentration. The tissue weight and the rate of protein synthesis in the gastrocnemius and soleus muscles were not affected by exercise. The results suggest that these changes in protein synthesis in the liver and small intestine may explain, at least partly, the atrophy of these organs which was observed after 7 d of exercise.

Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.

The anabolic actions of growth hormone and thyroxine on protein metabolism in Snell dwarf and normal mice.

The individual effects of GH and thyroxine (T4) on protein metabolism were determined in dwarf and normal mice in vivo. The hormone deficiencies of dwarf mice (low serum concentrations of GH and T4) resulted in decreased protein synthesis rates in skeletal muscle and liver, but no difference in synthesis rates in heart. The efficiency of synthesis (g protein/g RNA per day; KRNA) was lower in all three tissues in dwarf compared with normal mice, but effects on RNA concentration were not consistent; there was no change in muscle, a decrease in liver and an increase in heart. Treatment of dwarf mice for 9 days with either human GH or T4 caused increases in body weight and length. Protein synthesis rates were increased in muscle, liver and heart by either hormone, though much more so with T4 than GH. In muscle and liver both GH and T4 treatment resulted in an increased RNA concentration, but T4 treatment also increased KRNA. In heart, both GH and T4 increased KRNA with no change in RNA concentration. GH caused no significant changes in protein degradation rates so that growth rates were increased. T4 increased degradation rates so that there was no increased net growth in muscle or liver; in heart, T4 did induce increased growth despite the large increase in degradation rate. Tibial length was increased by both hormones; GH treatment of dwarf mice also increased cartilage sulphate incorporation on day 9, but T4 treatment did not, suggesting that bone growth is transient with T4 treatment. Normal mice showed no changes in growth or tissue protein metabolism in response to GH, but following T4 treatment there was increased protein turnover due to higher tissue RNA concentrations, although only heart growth was increased. Thus normal mice showed almost no net response to GH or T4, but dwarf mice showed a large response to both hormones. The response was different, however, in that GH caused concomitant increases in growth rates whereas T4 altered body tissue proportions.

RNA content of neurons in the ventromedial nuclei and lateral hypothalamic area relative to feeding status

The total RNA content of hypothalamic and cortex neurons in relation to the feeding status of adult male Wistar rats was studied. Experimental conditions including food deprivation (12 and 24 hours) and relative satiation (short-term refeeding, glucose or glycerol administration) changed in different ways the total RNA content of the neurons in the ventromedial hypothalamic nuclei (VMH) and in the lateral hypothalamic area (LHA) with respect to fasting or satiety. Only the long-term absence of food (24 hours) significantly increased the total RNA content of the VMH cells, while the RNA content of the LHA neurons significantly decreased in both the 12 and 24 hr fasted rats compared with those fed ad lib. The sixty minute free access to food after 12 or 24 hours of fasting fully reversed these changes. The short-term food intake significantly increased the RNA content of the LHA cells of the 12 and 24 hr fasted animals while the total RNA content of the VMH neurons significantly decreased only in the 24 hr fasted rats. The effect of glucose and glycerol administration on the RNA content of the LHA neurons (in 12 hr fasted rats) was similar to the effect of refeeding. One hour after giving glucose (1 g/kg b.wt.) or glycerol (300 mg/kg b.wt.) the total RNA content in the LHA neurons significantly increased. No changes in RNA content were observed in the neurons of the cortex when comparing the experimental and control rats. The results demonstrated the close relationship between the RNA content of the hypothalamic neurons and the feeding status. The contradictory changes of the total RNA content of the VMH and the LHA neurons during fasting or satiety reflected the recirpocal role of these hypothalamic structures in food intake control.

Distribution of DNA and RNA content in the bone marrow and alveolar macrophages of rats after subchronic inhalation of formaldehyde

The technique of flow cytometry was used to monitor the cell-cycle distribution of DNA and RNA in selected tissues of rats that were subchronically exposed to formaldehyde (HCHO) inhalation. Male Sprague-Dawley rats inhaled HCHO vapor concentrations of 0, 0.5, 3, or 15 ppm for 6 hr/day, 5 days/week, for up to 24 weeks. Simultaneous two-parameter measurements were made on a Phywe ICP 22 pulse cytophotometer by use of an acridine orange stain of the DNA and RNA of each cell sampled. No significant changes relative to the controls were determined in the percentage S and G2 + M phases of the DNA from sample tissues of the HCHO-exposed animals. Increases of 50 to 60% in the RNA content of G1 cells (RI) in the alveolar macrophages were seen after 1 week of exposure at all three HCHO doses. This effect was diminished after subchronic exposure. No HCHO-related effect was observed, though, in the RI of the rat bone marrow cells at any time point. However, the observed changes in RNA content were of rather limited magnitude relative to the cell-cycle perturbation induced by known cytotoxic agents in this and other flow cytometry studies. These modest and transient RI increases therefore probably did not reflect any significant effect on cell viability or cell proliferation in the affected tissues.

クロガシラガレイ稚子魚の成長に伴うたんぱく質量と核酸量の変化

Protein, DNA and RNA contents, together with the growth rates of body length and dry weight were measured for the cresthead flounder Limanda schrenki, during one month after hatching. The larvae showed no remarkable changes of external morphology in the first two weeks, started metamorphosis in the third week, and then into benthic phase in the fourth week. The maximum value of growth rates of body length and dry weight were observed in the second week. The changes in increasing rate of the protein showed the same tendency as that of dry weight. The values of protein increasing rate in the first week was almost equal to the growth rate of dry weight, while in the second week the former was lower than the latter. This result may be caused by the fact that some nonprotein materials accumulated in the second week. The pattern of changes in DNA content tended to be similar to changes in RNA content. The increasing rates of DNA and RNA were highest in the second week, and minimum rates were seen in the third week. Protein/DNA ratio indicated cell size, decreased rapidly during the first two weeks, whereas only in the third week did the ratio increase. The result indicated that cell proliferation occurred actively in the first two weeks and cell enlargement was taking place primarily in the third week. In the fourth week, there is less significant difference among the increasing rate of dry weight, protein, DNA and RNA.

Applications of flow cytometry to the structural and functional study of immune responsive cells.

Although flow cytometry is not yet widely used for diagnostic purposes, it provides a powerful tool for investigating structural and functional properties of immune-responsive cells. By far, the largest application of immunofluorescence is in identifying specific cell surface features thereby allowing the discrimination of lymphocyte subpopulations (phenotyping normal and malignant cells), for analysing complex receptors on neutrophils and monocytes, for detecting the appearance of new surface antigens induced by cell differentiation or activation. Besides this, the technique sets objective standards for appreciating cell activation (changes in RNA content, increased phagocytosis, DNA synthesis, changes in membrane fluidity, pH gradient changes). Computer processing of measurements allows rapid accurate studies of several properties simultaneously.

Changes in RNA content in neurons and perineuronal glia of the caudal vagus ganglion after a single maximal overloading.

Changes in RNA content in neurons and perineuronal glia of the caudal vagus ganglion after a single maximal overloading.

Denervation modulated changes in mouse skeletal muscle RNA concentration.

We have examined normal and denervated mouse skeletal muscle for histological evidence of changes in RNA concentration using acridine orange/nucleic acid fluorescence. In denervated muscle, cytoplasmic RNA concentration increases dramatically. The earliest increase is in the subsarcolemmal region, 7 days after denervation. By 28 days after denervation, the increase in RNA is distributed throughout the cytoplasm of the atrophic cells, non-atrophic cells show little or no increase. Prior to the increase in cytoplasmic RNA concentration, many myocyte nuclei exhibit enlarged nucleoli, indicative of active ribosomal RNA synthesis. Such active nucleoli are present throughout the 42 day study period. We conclude that both new RNA synthesis, and preferential accumulation of cytoplasmic RNA are prominent events in denervation atrophy. There is an ordered progression from nucleolar synthesis to subsarcolemmal accumulation to diffuse accumulation in atrophying cells. These observations complement biochemical findings of others which indicate an increase in both RNA and protein synthesis after denervation.

The differing responses of four muscle types to dexamethasone treatment in the rat

The glucocorticoid dexamethasone dramatically altered growth patterns in four muscle types, inducing atrophy of smooth and fast-twitch skeletal muscle, suppressing protein accumulation in slow-twitch muscle and enhancing growth in the heart. These differing responses were explained by steroid-induced changes in RNA content, protein synthesis and protein breakdown.

Changes in amount and synthesis of poly(A)- and poly(A)+ RNA in growing and resting cultures of Tetrahymena.

This investigation deals both qualitatively and quantitatively with the changes of RNA content and synthesis during the culture growth cycle of Tetrahymena. Affinity chromatography with an oligo(dT) column was used to separate poly(A)+ RNA from total RNA. The rates of synthesis of poly(A)- and poly(A)+ RNA were determined in terms of the incorporation of [5-3H]uridine. During the log phase, the cellular RNA and protein contents decreased steadily, whereas during the resting stage, both were constant. The extents of decrease of both fractions of RNA were essentially the same (54.4% and 50.6% for total and poly(A)+ RNA, respectively). Therefore, the relative contents of poly(A)+ RNA was constant from the beginning of the log to the resting stage (4.58%). The decrease in protein content, however, amounted to only 24.8%. Theoretically, a change in the age distribution during culture growth would cause a lower content of both fractions of RNA. The extents of the decrease in the rate of synthesis of both fractions were the same (75% and 79% for poly(A)- and poly(A)+ RNA, respectively). However, this reduction is so large that it cannot be solely the result of a shift of the age distribution of the cell population.

Changes in cytoplasmic RNA content of sympathetic neurons after synaptic block

Cytophotometry of RNA was carried out in neurons of the cranial cervical sympathetic ganglion of mature rabbits treated with different doses of the ganglion-blocker Dimecoline. After injection of the drug sharp fluctuations in the RNA content were observed in the cytoplasm of the neurons. The course of the fluctuations differed significantly depending on the dose of the drug injected, but it was similar in mono- and binuclear neurons. It is concluded from analysis of the character of differences found in the investigation that the results support the hypothesis of the leading role of synaptic processes in the formation of quantitative changes in the RNA content in the postsynaptic neuron. The results are evidence of increased functional activity of some ganglionic neurons after administration of different doses of Dimecoline both during and after the end of the block.

Scanning-integrating microdensitometric analysis of age-related changes in RNA content of cerebrocortical neurons in mice subjected to auditory stimulation

Cytophotometric analyses were made of auditory cortical RNA changes in 10-week and 40-week-old mice subjected to a 30-min interval of continuous noise stimulation (100 dB re 2 × 10 −5 N/m 2, 0.35 to 20 kHz). Treatment groups consisted of young and old noise-exposed mice killed at 0-, 1-, and 4-h intervals after cessation of noise stimulation. Corresponding groups of control mice were not subjected to noise stimulation. Quantification of neuronal RNA changes was made on an individual cell basis using scanning-integrating microdensitometry of azure B-stained brain sections. In the noise-stimulated groups we found that auditory cortical neurons from older mice synthesized and accumulated RNA to the same extent if not more than those of younger mice. The similarity in the pattern and extent of neuronal RNA augmentation in old and young mice after sensory stimulation suggests there is no marked age-related impairment in the capacity of cerebrocortical cells to synthesize RNA. Prior to stimulation we found that older mice had appreciably more neurons with very low RNA values compared to younger mice; older mice also exhibited a more rapid restoration of brain RNA toward control values at the 4-h postexposure interval.

In Vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells

Cytoplasmic RNA, isolated at various times after vaccinia virus infection, was translated in a message-dependent cell-free system prepared from rabbit reticulocytes. Supplementation of the system with calf liver tRNA specifically increased translation of viral RNA. Virtually all of the [ 35S]methionine-labeled viral proteins from infected cells that were detected by sodium dodecyl sulfate -polyacrylamide gel electrophoresis appeared to be synthesized in the cell-free system. When programmed with RNA extracted at 2 hr after infection, early viral proteins were made and formation of cellular proteins was diminished. Primarily late proteins were synthesized using RNA extracted at 4 or more hr after infection, suggesting that the switch in protein synthesis is regulated principally by changes in RNA concentration rather than by modification of the translation apparatus of the cell. However, the vaccinia virus-mediated inhibition of host protein synthesis that occurred in the presence of actinomycin D was not associated with a decrease in translatable cellular mRNA. Immediate early RNA and early RNA were obtained by infecting cells in the presence of inhibitors of protein and of DNA synthesis, respectively. Analysis of the in vitro translation products did not reveal a class of early genes that require protein synthesis for expression. On the contrary, seven polypeptides, of which a 28,000-dalton species was most prominent, were synthesized in relatively greater amounts with immediate early RNA than with early RNA. All early and late mRNA species appear to be polyadenylylated, and a correlation between RNA sedimentation and molecular weight of translation product was obtained.

Cytochemical comparison of two kinds of experimental hypoxia: Changes of RNA content per cell in cerebellar and cerebral cortex neurons and perineuronal glia

Cytochemical comparison of two kinds of experimental hypoxia: Changes of RNA content per cell in cerebellar and cerebral cortex neurons and perineuronal glia