Dietary nucleotides correct plasma and liver microsomal fatty acid alterations in rats with liver cirrhosis induced by oral intake of thioacetamide

BACKGROUND/AIMS: Dietary nucleotides modulate a number of metabolic processes, including long-chain polyunsaturated fatty acid metabolism. In this study, we evaluated the effect of dietary nucleotides on plasma and liver microsomal fatty acid profiles in a rat model of liver cirrhosis induced by oral intake of thioacetamide. METHODS: Fifty-four female Wistar rats were assigned to one of the following groups: rats in the thioacetamide group (n=45) were given 300 mg thioacetamide/l in their drinking water for 4 months, and rats in the control group (n=9) received water during the same period. After 4 months of treatment, 9 rats in each group were killed. The remaining rats in the thioacetamide group were divided into two new groups, and the animals in each were allowed to recover for 1 or 2 weeks on either a nucleotide-free diet or the same diet supplemented with 50 mg of each of the following: AMP, GMP, CMP, IMP and UMP per 100 g diet. RESULTS: Saturated (mainly stearic acid), monounsaturated, and n-6 long-chain polyunsaturated fatty acids (mainly arachidonic acid), and also the unsaturation index decreased in plasma of rats with experimental cirrhosis. Administration of the diet supplemented with nucleotides to thioacetamide-treated rats corrected plasma levels of saturated, n-6 long-chain polyunsaturated fatty acids and the unsaturation index. In liver microsomes, the cirrhotic rats showed lower levels of protein and higher levels of palmitic, oleic, linoleic and arachidonic acids. Protein concentrations and levels of all the above-mentioned fatty acids were corrected with the nucleotide-enriched diet. CONCLUSIONS: Dietary nucleotides contribute to correcting plasma and liver microsomal fatty acid alterations in rats with liver cirrhosis induced by chronic oral administration of thioacetamide.     

Faster synthesis and slower degradation of liver protein during developmental growth

A study is presented of the liver protein gain during the early stages of postnatal development. Fractional rates of protein synthesis and degradation were determined in vivo in livers of 4-day-old mice. At this age, liver protein accumulated at a rate of 18% per day. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was extimated as the balance between synthesis and accumulation of stable liver proteins, or from the disappearance of radioactivity from liver protein previously labelled by the administration of NaH14CO3. We found that the neonatal livers: (1) synthesize 139% as much protein per unit time and unit mass as adult tissue, which is accounted for by a higher ribosome concentration (synthesis per mg of RNA was the same); (2) retain 39% of the newly synthesized protein as stable liver components (compared with 48% in adult mice); (3) degrade protein at 56% of the rate in the adult liver. This lower rate of degradation is quantitatively the most significant difference between the growing and non-growing liver.     

Effect of starvation of the distribution of free and membrane-bound ribosomes in rat liver and on the content of phospholipid and glycogen in purified ribosomes

The distribution of free and membrane-bound ribosomes in liver in response to starvation has not been clearly defined. An investigation has been made of the effects of starvation on the content of DNA, RNA, protein, phospholipid and glycogen in rat liver, on the distribution of free and membrane-bound ribosomes, and on the content of phospholipid and glycogen in free and bound ribosome fractions. The results indicate that starvation can produce up to a 50% reduction in hepatic ribosomes without altering either the fraction of rRNA relative to the total RNA or the distribution of free and membrane-bound ribosomes. In addition, the degree of contamination of isolated ribosomes with membranous material does not fluctuate with changes in the nutritional status of the animal. The results suggest that the relative capacities for protein synthesis among the two ribosome compartments are maintained during the early stages of starvation. Further, co-sedimentation with glycogen is not responsible for the presence of membranous materials in purified ribosomes.     

Effect of cholingeric drugs on methadone-induced catalepsy and stereotypies in rats treated chronically with methadone

The rate of synthesis of transfer RNA in suspension cultures of Chang's liver cells, has been examined in the presence of various inhibitors of protein synthesis with different modes of action. Inhibitors of polypeptide chain elongation such as cycloheximide and emetine stimulated the rate of synthesis of transfer RNA at concentrations that inhibited protein synthesis by 60-90%. Trichodermin, an inhibitor of the elongation and termination steps in protein synthesis, had as effect similar to that of cycloheximide and emetine. On the other hand verrucarin, an inhibitor of initiation, and puromycin, an analogue of the aminoacyl terminus of tRNA, had little effect of the synthesis of transfer RNA at low concentrations. At high concentrations these compounds inhibited transfer RNA synthesis. Inhibitors of protein synthesis can be divided in two groups based on their effect on the polysom pattern. The first group increased the number of large polysomes, while verrucarin and puromycin lead to a break down of large polysomes and to an accumulation of monosomes and small polysomes. Thus, there appears to be a correlation between the effect of these inhibitors of protein synthesis on the aggregational state of the polysomes and their effect on the rate of transfer RNA synthesis.     

Subunit structure of isomeric chondroitin sulfates from normal human urine

Two temperature-sensitive mutants of Chlamydomonas reinhardii Dangeard which are defective in protein synthesis were examined. Both show breakdown of their polysomes at the restrictive temperature into monosomes which do not contain fragments of mRNA. Many of the ribosomes still contain nascent peptides able to react with puromycin. The polysome breakdown involves only cytoplasmic (80S) ribosomes and is prevented or reversed when ribosome translocation is inhibited with cyloheximide.     

Hematopoietic bone marrow hyperplasia: correlation of spinal MR findings, hematologic parameters, and bone mineral density in endurance athletes

To clarify the effects of obstructive jaundice on the liver, sequential changes of hepatic energy charge, the concentrations of adenine nucleotides and malondialdehyde, DNA synthesis rate, and histology of the liver were examined on the day before and Days 1, 2, 4, 7, and 14 after biliary obstruction in rats and compared with those of sham-operated controls. Foci of necrotic hepatocytes were present on Days 1 and 2 and mitoses of the hepatocytes were frequently observed with a peak on Day 2 in the jaundiced liver. Marked proliferation of bile ductules were subsequently observed on Days 7 and 14, resembling biliary cirrhosis. The DNA synthesis rate was significantly activated after bile duct obstruction with its peak on Day 2, more than nine times higher than the control value and returned to the control level on Day 14. Hepatic ATP concentration and energy charge gradually declined with prolonged jaundice and significantly lower levels persisted after Day 7 compared with the controls. The malondialdehyde level in the jaundiced liver gradually increased and became significantly higher on Day 14. We conclude that obstructive jaundice decreases hepatic energy charge and increases the lipoperoxide level. In the initial stage of obstructive jaundice, the hepatocytes proliferate associated with activated DNA synthesis probably to compensate hepatic damage; however, prolonged obstructive jaundice induces functional hepatic injury possibly necessitating biliary drainage.     

Protein synthesis in vivo in rats fed on lipid-rich liquid diets

Changes in tissue composition and protein synthesis ratio were studied in the major tissues of the body in young rats fed on lipid-rich, isonitrogenous purified liquid diets, a convenient method for inducing voluntary overfeeding under controlled nutritional conditions. Overfed rats showed faster growth induced by the energy excess. Analysis of tissue composition (protein, DNA and RNA contents) revealed that growth was due mainly to tissue hyperplasia in which protein and DNA contents increased in parallel. Fractional protein synthesis ratio measured in vivo by the flooding-dose method of phenylalanine showed a marked increase in all tissues. This change could be attributed to an increase in the ribosomal activity for protein synthesis in most tissues. Therefore, our results indicate that addition of a supplementary energy source (as lipids) to a well-balanced diet improves growth and protein synthesis in growing rats.     

Differential effects of chronic ethanol consumption on hepatic mitochondrial and cytoplasmic ribosomes

The effects of chronic ethanol consumption on the properties of mitochondrial and cytoplasmic ribosomes were investigated in rat liver. Sedimentation properties of purified mitochondrial (55S) and cytoplasmic (80S) ribosomes were determined by analyses on sucrose density gradients. Mitochondrial ribosomes from control animals moved further in the gradients than did those isolated from ethanol-fed rats, which suggests that ethanol ribosomes have a lower molecular weight. In addition, mitochondrial from ethanol-fed animals contained a lower percentage of ribosomes present as the intact monosome, suggesting that ethanol may have an effect on the stability of the functional mitochondrial ribosomes. This was confirmed by the presence of the larger 39S subunit in preparations from ethanol-fed animals. No such ethanol-related alterations were seen with cytoplasmic ribosomes. The protein composition of mitochondrial cytoplasmic ribosomes was investigated using two-dimensional gel electrophoresis, followed by two-dimensional densitometry. As indicated by differences in protein staining intensity, ethanol consumption seemed to alter the concentration of seven mitochondrial ribosomal proteins. In contrast, no such changes were observed in the protein pattern from cytoplasmic ribosomes. Observations in this study provide for the possibility that alterations in the amounts of selected proteins in the mitochondrial ribosome lead to impaired assembly of the ribosome. These ethanol-related structural changes may be responsible for the decreased activity of mitochondrial ribosomes that results in impaired hepatic mitochondrial protein synthesis (W.B. Coleman and C.C. Cunningham, Biochim. Biophys, Acta 1058:178-186, 1991). Furthermore, this study reemphasizes the increased susceptibility of the hepatic mitochondrial translation system, compared with the cytoplasmic system to chronic ethanol consumption.     

Effects of methylation of the beta-galactosidase genome upon in vitro synthesis of beta-galactosidase

A template DNA from phage lambdah80dlacp5 coding for the in vitro synthesis of beta-galactosidase was used to study the effect of DNA methylation by the alkylating agent, dimethyl sulfate (DMS). Increasing the levels of DMS up to 50 mM concentration in the incubation medium led to an increase of DNA methylation. When incubated for 10 min at 37 degrees C, 3-4% Of nucleotides were methylated. The increase was linear to about 0.6% nucleotide methylation level. A higher yield was obtained at 37 degrees C incubation temperature than at 20 degrees C. Methylation of lambdah80dlacp5 DNA alone without methylation of other factors in the incubation mixture caused inhibition of the synthesis of beta-galactosidase in vitro. Increasing levels of DNA methylation caused greater inhibition of the newly synthesized enzyme activity. Total protein and RNA synthesis was inhibited by the methylated DNA to a much lesser extent than the inhibition of enzyme activity. When the level of nucleotide methylation was 0.74%, only 2% of enzyme activity remained, but total protein and RNA synthetic activities were found to be 72% and 44%, respectively.     

Comparison of the temperature sensitivity of protein synthesis by cell-free systems from liver of rat and skate (Raja ocellata)

Studies were undertaken to determine the component(s) responsible for the temperature optimum characteristic of the protein-synthesizing system from skate and rat. 1. The macromolecular constituents of rat and skate liver ribosomes are compared. The number of ribosomal proteins is similar in the two species, although most proteins display different electrophoretic mobilities on polyacrylamide gels. The RNAs from the small subunit of skate and rat have similar sedimentation coefficients; however, the RNA from the large subunit of skate ribosomes appeared to be slight smaller than the comparable RNA from the rat. 2. Ribosomes from either rat or skate were capable of supporting poly(U)dependent polyphenylalanine synthesis with soluble factors from either species. 3. Maximal leucine incorporation directed by endogenous mRNA occurred at 35--40 degrees C with post-mitochondrial supernatant from the rat liver and at 20--30 degrees C with that from skate liver. 4. The characteristic temperature sensitivity of protein synthesis was dependent upon the source of cell sap and independent of the source of ribosomes. 5. Elongation factor 1 from both the rat and skate exhibited maximum activity at approx. 30 degrees C. 6. Phenylalanyl-tRNA synthetase from skate liver showed maximum activity at 30 degrees C while that from rat was maximally active at 37 degrees C. The rat enzyme, however, was active at 0--10 degrees C, at which temperature protein synthesis in the reconstructed rat system is virtually absent. 7. The protein-synthesizing capacity of the reconstituted system at various temperatures was closely correlated with the activity of Elongation factor 2 (translocase). Elongation factor 2 from rat liver displayed an optimum at 30 degrees C and lost all activity below 10 degrees C, while this same factor from skate liver showed an optimum at 20 degrees C and significant activity below 10 degrees C. At this low temperature the reconstituted skate liver system continued to exhibit the ability to synthesize protein. These studies suggest that Elongation factor 2 is the component responsible for determining the temperature at which the protein-synthesizing system displays its characteristic maximum activity.     

Soybean protein suppresses hepatic lipogenic enzyme gene expression in Wistar fatty rats

The effects of dietary soybean protein on lipogenic enzyme gene expression in livers of genetically fatty rats (Wistar fatty) have been investigated. When Wistar fatty rats and their lean littermates (7-8-wk old) were fed a casein or soybean protein isolate diet containing hydrogenated fat (4% hydrogenated fat plus 1% corn oil) or corn oil (5%) for 3 wk, the hepatic messenger RNA concentrations and activities of lipogenic enzymes were significantly lower in rats fed soybean protein than in those fed casein, regardless of genotype or dietary fat. The conversion rates of thyroxine to triiodothyronine by liver microsomes and plasma triiodothyronine concentrations were lower in the fatty rats than in the lean rats and were significantly greater in rats fed soybean protein than in those fed casein. Conversely, plasma and liver triacylglycerol concentrations were lower in soybean protein-fed fatty and lean rats than in those fed casein. The body weight was less in the fatty rats fed soybean protein than in those fed casein after 3 wk of feeding. Moreover, dietary polyunsaturated fatty acids suppressed lipogenic enzyme gene expression in the lean rats but did not in the fatty rats. Dietary soybean protein appeared to be useful for the reduction of obesity.     

Cyclooxygenase-2 expression in human esophageal carcinoma

RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (-17, -14, -13, and -11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238-11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides -17 and -11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides -11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.     

Influence of Ukrain on DNA, RNA and protein synthesis in malignant cells

3H labelled thymidine, uridine and leucine were used to evaluate the synthesis of DNA, RNA and proteins in malignant cells and normal cells incubated with Ukrain in different concentrations. Compared with the controls, the inhibiting effects of Ukrain are demonstrated in guinea pig hepatocytes, CIL hepatocytes, human tonsil cells, two murine lymphomas, murine myeloma, Yoshida cells, two HeLa strains, EsB- and EB- (murine) lymphomas. YAC-1, P815 and human WiDr cells. Ukrain inhibits the DNA, RNA and protein synthesis in malignant cell lines at relatively high concentrations and to a small extent in normal cells.     

A muscle hypertrophy condition in lamb (callipyge): characterization of effects on muscle growth and meat quality traits

The present experiment was conducted to determine the effect of the callipyge phenotype on traits affecting muscle growth and meat tenderness. Dorset wethers (N = 40) that were either carriers or non-carriers were fed grain and slaughtered at 169 d of age. Callipyge phenotype did not affect (P > .05) slaughter weight, hot carcass weight, or weights of the heart, spleen, viscera, kidney-pelvic fat, head, and pelt; however, callipyge lambs had a higher dressing percentage and lighter lungs, liver, and kidneys (P < .01). Callipyge lambs had reduced fat thickness and marbling score and higher leg scores and longissimus area (34%). Adductor (30%), biceps femoris (42%), gluteus group (31%), longissimus (32%), psoas group (20%), quadriceps femoris (18%), semimembranosus (38%), and semitendinosus (26%) weights were higher in the callipyge phenotype (P < .01); however, phenotype did not affect (P > .05) weights of infraspinatus or supraspinatus. Longissimus pH and temperature declines were not affected (P > .05) by phenotype. Longissimus myofibril fragmentation index was lower at 1 (27%), 7 (35%), and 21 (37%) d postmortem and Warner-Bratzler shear force was higher at 1, 7, and 21 d postmortem in the callipyge phenotype (P < .01). Shear force values of callipyge lambs at 21 d postmortem tended to be greater (P = .12) than shear force values of non-carriers at 1 d postmortem . Activities of calpastatin (83%) and m-calpain (45%) were higher in the callipyge (P < .01); however mu-calpain activity was not affected (P > .05). Longissimus and semitendinosus RNA concentration, DNA content, RNA content, protein content, and the RNA:DNA ratio were higher (P < .05), but DNA concentration, protein concentration, and protein:DNA were not affected in the callipyge phenotype. The higher calpastatin activity associated with callipyge suggests that protein degradation may be reduced in the live animal. Additionally, the increased muscle DNA content associated with the callipyge phenotype suggests an increase in satellite cell proliferation, and results in an increased capacity of skeletal muscle to accumulate and maintain myofibrillar protein. These results suggests that both reduced rate of protein degradation and higher capacity for protein synthesis are consequences of the callipyge condition.     

Protein synthesis and degradation change rapidly in response to food intake in muscle of food-deprived mice

The short-term changes in muscle protein synthesis and degradation after food intake are unclear. We investigated muscle protein metabolism after food intake in mice that were starved for 18 h and refed for 1 h. Protein synthesis activity was estimated by the polysome profiles, and protein degradation was estimated by plasma N tau-methylhistidine (MeHis) concentration, reflecting translational activity and myofibrillar protein degradation, respectively. MeHis is an index of myofibrillar protein degradation because it is not reused for protein synthesis and it is not metabolized. Stimulation of protein synthesis (polysome profile) and the reduction of protein degradation (plasma N tau-methylhistidine concentration) were observed immediately after feeding began. Protein synthesis returned to the prefeeding level by 6 h after refeeding, whereas protein degradation remained at a low level. The decreased plasma MeHis concentration after refeeding was not due to a decrease in MeHis release from muscle cells and an increase in the free MeHis pool size, because the changes in free MeHis concentration in muscle were similar to that of plasma. Plasma insulin concentration immediately rose with feeding and it returned to the prefeeding level by 3 h after refeeding. These results suggest that responses of postprandial protein metabolism are very rapid and that protein synthesis is regulated by insulin, whereas degradation is regulated by insulin and other dietary factors. Thus the ability of skeletal muscle to use nutrients more effectively by stimulating protein synthesis and reducing protein degradation may cause the accelerated rate of protein accretion in skeletal muscle during the short postprandial period.     

Muscle growth in two genetically different lines of swine

Skeletal muscle growth in two genetic lines of pigs that differed in total muscle content was studied at live weights of 23, 45, 68, 91, 104, 118 kg. Total physically separable muscle and cross-sectional area of the longissimus dorsi muscle were greater in the muscular than in the obese genetic lines. Above 45 kg, animals in the muscular genetic line had less total separable fat than animals in the obese line, but the two lines did not differ in total physically separable fat at 23 and 45 kg live weight. Hence, these two genetic lines may differ in weight at which maturity is reached as much as in inherent propensity for obesity. Longissimus muscle form the muscular line had more water, less protein, and less lipid than longissimus from the obese line. DNA and RNA concentration, total DNA and RNA content, and RNA/DNA ratio of the pituitary and liver did not differ between the two genetic lines. Above 68 kg, longissimus from the muscular line had higher DNA and RNA concentrations than longissimus from the obese line; this difference did not exist between 23 and 68 kg. RNA/DNA ratio of the longissimus muscle was greater and protein-to-DNA and protein-to-RNA ratios in longissimus were lower in the muscular than in the obese line. Total DNA content of physically separable muscle increased 2.0 (obese) to 2.7 (muscular)-fold between 23 and 118 kg; hence, number of muscle nuclei increases during growth. Total DNA content of physically separable muscle was greater in the muscular than in the obese line and was the measurement most highly related to total muscle content.