Perfusion in situ with tritium oxide to measure hepatic lipogenesis and lipid secretion. Normal and orotic acid-fed rats.
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Animals↗
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[The effect of active substances on nerve fiber regeneration in nerve tissue cultures].
Explants of the ganglion trigeminale from chick embryos and hippocampi from embryonal rats were incubated in maximowchambers with semisynthetic media. The different parts of nervous tissue were influenced experimentally by addition of biological extracts and by substances with known composition. The regeneration of nerve fibers was investigated by the index of growth. The growth index was calculated from the ratio of nerve fibre index of the test cultures to that of the influenced cultures. Under these conditions biological extracts enhanced the growth of nerve fibres. In the same way the growth of nerve fibres was statistically significantly stimulated by substances with known composition of aminoacids, orotic acid, sodium orotate and cyclic monophosphates. A stimulating effect of cyclic guanosinmonophosphate seems to exist only in CNS explants and only in young fetal rats and consists in an increased migration and proliferation of cells as well as in the formation of fibres from neuroblasts. The investigations gave strong evidence for the in vitro testing to be very useful in studies of nerve fibre regeneration. However the choice of suitable reference systems, suitable quantitative parameters, optimal concentration and periods of application of effective substances and the age of the animals are of fundamental importance for the evaluation of the results. Experiments regarding the stimulation of the differentiation of neurons and the growth of nerve fibres are of practical clinical and therapeutical interest.
Animals↗
[Problems relating to the use of radioactively labelled bacteria in animal experiments. 3. Distribution of activities in mice, following parenteral administration of Pasteurella multocida labeled with various radionuclides].
When radioactively labelled fully developed Pasteurella (P.) multocida antigens were parenterally applied to mouse, through intravenous, intraperitoneal or subcutaneous routes, activity distribution was found to depend both the technique of application and the radionuclide used. This has given rise to the conclusion that activity distribution could be equated with whole-germ distribution only under certain conditions. Experimental antigen distribution, accordingly, reflected a complex process in which transport, location, and metabolism of bacteria had to be considered as being in conflict with the given host organism. Iodine labelled 125 was split off bacteria at relatively high rate and accumulated in the thyroid gland. When 59Fe was used, interpretation of distribution was rendered more difficult by propagation of iron through the organs of the host. Relatively favourable data were obtainable from the use of 3H or 14C which could be used in the form of nucleic acid precursors (orotic acid) in labelling Pasteurella. Intraperitoneal and intravenous forms of administration were found to be the same for intra-organic activity distribution. Retention of activity in liver, spleen, and lung was recordable and established from all the reviewed techniques for the application of P. multocida complete antigen. While maximum concentrations were measured between the first and third days, activities still were detectable after 14 days (period under review). Relatively high radioactivity was recordable from the digestive tract, within short time from the use of the parenteral procedures tested. This seemed to suggest passage of complete or incomplete P. multocida antigen through the mucous membranes of the gastro-intestinal tract.
Animals↗
The synthesis of ribonucleic acid in vivo in the nuclei of rat brain fractionated by zonal centrifugation.
1. Radioactive orotic acid, uridine and adenosine were administered to rats by intracisternal injection. The effects of the size of the dose, the specific radioactivity and time on the incorporation into the RNA of unfractionated nuclei of brain tissue were examined to establish appropriate conditions for studies of the relative activities in vivo of the various sorts of brain nuclei fractionated by zonal centrifugation. Uridine is incorporated more efficiently than either orotic acid or adenosine. 2. With [(3)H]uridine as precursor the astrocytes in zone (II) contain the highest radioactivity except at the beginning of the experiment when the neuronal nuclei of zone (I) are more highly labelled. This fraction utilizes [(14)C]orotic acid more readily than the nuclei of zone (II). The astrocytic nuclei of zone (III) show a general resemblance to those of zone (II). Considerable differences between the incorporations into the two types of oligodendrocyte nuclei in zones (IV) and (V) are observed. 3. The relative synthetic activities of the major types of brain nuclei in vitro and in vivo are discussed.
Adenosine↗
Effect of milk constituents on hepatic cholesterol-genesis.
Two preparations active in reducing hepatic cholesterol biosynthesis were isolated from bovine skim milk. One of the inhibitors was in the dialysate and was identified as orotic acid (OA). The other inhibitor, present in the retentate, was not identified. Orotic acid appears to act by inhibiting cholesterol biosynthesis before the formation of mevalonate, whereas the retentate inhibitor exerts its effect beyond the formation of mevalonate in the biosynthetic pathway. Human milk also inhibited the incorporation of both labeled acetate and mevalonate into cholesterol by rat liver. Orotic acid was not detectable in human milk samples employed in this study. Administration of [6-14C]orotate to rats revealed its conversion to uracil in the liver. Subsequent work demonstrated that uracil had inhibitory activity on hepatic cholesterol biosynthesis similar to that of orotate when incubated with rat liver slices.
Acetates↗
AN OROTIC ACID-INDUCED, ADENINE-REVEREED INHIBITION OF HEPATIC LIPOPROTEIN SECRETION IN THE RAT.
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Adenine↗
Fetal fuels. V. Ketone bodies inhibit pyrimidine biosynthesis in fetal rat brain.
Ketonemic states complicating late pregnancy are accompanied by lower brain weights in the newborn. Potential mechanisms whereby ketone bodies might inhibit cell proliferation were therefore examined in the fetal rat brain slice by measuring their impact on the de novo pathway for pyrimidine biosynthesis. DL-beta-hydroxybutyrate (10.8 mM) and acetoacetate (5.4 mM) were both found to diminish the incorporation of NaH14CO3 into [14C]UMP by 30%. This effect was similar in fetal tissues from fed and 48-h starved mothers. Graded concentrations of DL-beta-hydroxybutyrate (1.4-43.2 mM) resulted in a progressive inhibition that could not be explained either by isotope dilution consequent to ketone body oxidation or by a generalized inhibition of protein synthesis. The inhibition was not reversed with 10 mM glutamine, the principal nitrogen substrate for de novo biosynthesis of pyrimidines. When the conversion of orotic acid into UMP was blocked with 6-azauridine, DL-beta-hydroxybutyrate (10.8 mM) inhibited the incorporation of NaH14CO3 into orotic acid by 28%. By contrast, maximally inhibitory concentrations of this ketone body (43.2 mM) had no effect on the incorporation of [6-14C]orotic acid into [14C]UMP. Is is concluded that ketone bodies inhibit the de novo biosynthesis of pyrimidines in fetal brain slices and that they do so at a site proximal to orotic acid formation.
Animals↗
A biochemical investigation on chicken gout observed in the Marmara region in Turkey.
Poultry breeding has economical importance for the people living in Bursa and surrounding villages. Similar to some sporadic disease, gout can also be reason for production loses and death in laying and broiler chickens. Gout cases have been observed in some pens of a big poultry company. In this study 20 healthy control and 40 sick Studler Iso-Brown chickens were used as research materials. Blood were taken from heart by syringe to plastic tubes with EDTA. Plasma were collected and analyzed for total protein, uric acid, bicarbonate, vitamin A, calcium and orotic acid. The blood levels of healthy and sick groups were; total protein % 4.60-5.98 gr, uric acid % 10.19-38.09 mg, bicarbonate 27.37-35.73 mEq/l, vitamin A % 42.20-35.55 mcg, calcium % 8.38-8.25 mg and orotic acid % 1.42-1.97 mg, respectively. Statistical analysis was done by t test. There were statistical importance for total protein, bicarbonate (p less than 0.01) and for uric acid (p less than 0.001) differences. Feeding program analysis was done and a feeding disturbance was determined. Chicken in gout observed pens have fed with chicken developing feed for two weeks earlier. For this case diet was the reason of gout.
Animals↗
A rapid and simple screening method for detection of orotic aciduria by capillary zone electrophoresis.
A rapid and selective capillary zone electrophoretic method for screening of patients with orotic aciduria is described. The method is based on direct measurement of orotic acid in untreated urine. Total analysis time is 5 min with the limit of quantification of 10 mumol/l. Selectivity of the method is given by the use of a low pH of background electrolyte at which practically no interferences from native urine appeared, as well as by using a fast scanning detector which enables the identification of orotic acid via its characteristic UV spectra. This method can be used for quantitative assessment of orotic acid present in urine at physiological conditions after an ion exchange chromatography-clean up and preconcentration procedure. Urine samples containing orotic acid at pathological concentrations (typically more than 1 mol/mol creatinine) could be successfully analysed. The method described was applied to urine specimens collected from both healthy volunteers and patients with orotic acidurias of various origin.
Acidosis↗
Effects of Cycloheximide upon Formation of Ribonucleic Acid Cytidylic and Uridylic Acids.
Concentrations of cycloheximide as low as 3 mug/ml inhibited incorporation of labeled orotic acid or uridine into RNA cytidylic acid of soybean (Glycine max) hypocotyl sections. Even lower concentrations of this well known protein synthesis inhibitor interfered with conversion of labeled cytidine into RNA uridylic acid. Both cycloheximide and puromycin inhibited absorption of (3)H-phenylalanine and its incorporation into protein, but puromycin did not significantly affect the labeling patterns of RNA cytidylic and uridylic acids when orotic acid-6-(14)C was fed. Results give further support to the hypothesis that cycloheximide inhibits the interconversion of uridine and cytidine nucleotides, presumably by acting as a glutamine antagonist in the glutamine-dependent reaction catalyzed by cytidine triphosphate synthetase.
Journal Article↗
An earlier proliferative response of hepatocytes in gamma-glutamyl transferase positive foci to partial hepatectomy.
One of the hallmarks of initiated hepatocytes is their resistance to several hepatotoxins. This property forms the basis for their selective growth under conditions which are inhibitory to the non-initiated hepatocytes. Selective growth of initiated hepatocytes also occurs, albeit at a low level, in initiated rat liver without exposure to any known promoting regimen and/or in the absence of any known selective pressure to which initiated hepatocytes can possibly be resistant. This latter phenotypic property of initiated hepatocytes was further characterized by comparing the kinetics of response of hepatocytes in gamma-glutamyl transferase positive foci and in the surrounding liver to 2/3 partial hepatectomy both in the presence and in the absence of a promoting regimen. Male Fischer 344 rats (130-150 g) were initiated with a single dose of diethylnitrosamine and 1 week later they were placed on either a semi-synthetic basal diet or a promoting diet containing 1% orotic acid. Partial hepatectomy was performed 15 weeks after initiation and animals from both groups were killed at 12, 16, 20, 24, 30, 36, 48, 72 or 96 h after operation. Each animal received a pulse of 3H-labelled thymidine 1 h prior to killing. Autoradiographic studies revealed that hepatocytes in gamma-glutamyl transferase positive foci in the livers of rats fed the basal diet were significantly labelled at 16 h post-partial hepatectomy while surrounding hepatocytes were still virtually quiescent (LI 12.7 +/- 4.7 versus 1.2 +/- 0.5%, respectively). Higher labelling index in foci compared to the surrounding liver was also seen at 20 h post-PH (36.9 +/- 2.6 versus 21.5 +/- 2.4). Similar earlier response of hepatocytes in gamma-glutamyl transferase positive foci was also seen in initiated rats exposed to dietary orotic acid. In addition, orotic acid treatment appears to have imposed a slight delay on the entry of hepatocytes in the surrounding liver into 'S' phase and thereby enhancing the differential of growth response between these two populations.
Animals↗
Stimulating actions on ribonucleic acid biosynthesis of aconitines, diterpenic alkaloids of Aconitum roots.
Mesaconitine (MA) significantly stimulated the incorporation of 5-[3H]-orotic acid into liver nuclear RNA 16 h after its administration. MA exhibited the strongest activity among the aconitine alkaloids. This stimulatory effect of MA was inhibited by actinomycin D, but the incorporation ratio of 5-[3H]orotic acid in the combined treatment group with MA and actinomycin D was significantly higher than that in the single treatment group with actinomycin D. MA also increased the incorporation of 5-[3H]orotic acid into polysomal RNA in mouse liver. When MA was added to the RNA polymerase (EC 2.7.7.6) preparation obtained from rat liver, the increase of the enzyme activity was weak. While RNA polymerase preparation from the liver of rats, which were previously treated with MA, elicited increased incorporation of [3H]cytidine monophosphate into RNA as compared with that from the livers of normal rats. Accumulated data indicate that aconitine alkaloids, in particular MA, accelerate liver RNA synthesis mainly by the increase of RNA polymerase.
Aconitine↗
Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo.
The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: (a) demonstration of the incorporation of [(14)C]NaHCO(3), [(14)C]carbamylaspartate, and [(14)C]orotic acid into uridine nucleotides; (b) the isolation of [(14)C]orotic acid when [(14)C]NaHCO(3) and [(14)C]carbamylaspartate were used as precursors; (c) the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and (d) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts.Regulation of the activity of the orotate pathway by end product inhibition was demonstrated in the intact cells of excised roots by measuring the influence of added pyrimidine nucleosides on the incorporation of [(14)C]NaHCO(3) into uridine nucleotides. The addition of either uridine or cytidine inhibited the incorporation of [(14)C]NaHCO(3) into uridine nucleotides by about 80%. The observed inhibition was demonstrated to be readily reversible upon transfer of the roots to a nucleoside-free medium. Experiments employing various radiolabeled precursors indicated that one or both of the first two enzymes in the orotate pathway are the only site(s) of regulation of physiological importance.
Journal Article↗
Transit and sorting of apolipoprotein B within the endoplasmic reticulum and Golgi compartments of isolated hepatocytes from normal and orotic acid-fed rats.
Apolipoprotein B (apoB) secretion by isolated rat hepatocytes was dependent on addition of oleate to the incubation medium and inhibited in hepatocytes isolated from livers of orotic acid-fed rats (OA hepatocytes). To investigate the intracellular transit of newly synthesized apoB under different conditions, normal hepatocytes (with or without oleate) or OA hepatocytes (with oleate) were incubated with [35S]methionine, and subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, cis-Golgi, and trans-Golgi and membrane and lumenal contents from these) were isolated at intervals. The specific activities and pool sizes of apoB100 and apoB48 were determined. The observations indicate that there are several points at which intracellular transit of apoB is regulated. Newly synthesized apoB is either translocated to the lumen of the rough endoplasmic reticulum or remains membrane bound and is degraded. The lumenal apoB is either retained and degraded or transferred to the Golgi lumen and secreted. In OA hepatocytes degradation of the membrane-bound form of apoB is inhibited, and the protein accumulates in the trans-Golgi membranes. Although apoB is translocated to the lumen of the rough endoplasmic reticulum in OA hepatocytes, it is not packaged with lipid and is transferred to the Golgi lumen only slowly.
Animals↗
Acid-soluble purine and pyrimidine derivatives in cow's milk produced on normal feed and almost protein-free-feed with urea as the main nitrogen source.
In two groups of Ayrshire cows (group I normal feeding with protein, group II got mostly urea as N-source), the TCA-soluble purine and pyrimidine derivatives of the milk were determined quantitatively. The O.D. [U/I] at pH 7-5 and 260 nm was higher in group II than in group I. The difference in N-nutrition of the animals has no influence on the composition of the purine and pyrimidine fractions investigated, especially with regard to orotic acid, uric acid, and CMP. At early stages of lactation the contents of CMP and uric acid were relatively high, but decreased with longer time of lactation. In opposition to this the orotic acid content increased in the same period. These tendencies could be registered in both groups. Besides CMP, uric acid, and orotic acid the minor nucleotides, NAD, AMP, GMP, UMP and others have been determined quantitatively. Their maximum has been found at the beginning of the lactation period and, in general, their contents were higher in group II than in group I.
Animal Feed↗
The relationship between urea and pyrimidine de novo synthesis in ruminant liver.
The influence of ammonia and inhibition of the urea cycle by norvaline on orotic acid synthesis in five Polish Merino ewes was investigated. The sheep were exposed to 60 min of successive infusions of 0.9% NaCl, NH4Cl (20 mumol kg-1 min-1), and NH4Cl with DL-norvaline (10 mumol kg-1 min-1) solutions administered to the mesenteric vein. The average rate of orotic acid output, urea output and ammonia uptake in the liver in control conditions (infusion of 0.9% NaCl) was: 1.09 nmol g-1 min-1, 0.97 mumol g-1 min-1 and 0.94 mumol g-1 min-1, respectively. Ammonia significantly stimulated the orotic acid output, urea output and ammonia uptake. At the same time liver uptake of glutamic acid, alanine and glycine increased. Norvaline as a competitive inhibitor of ornithine transcarbamylase depressed citrulline release and urea output in the liver, and elevated, although not significantly, the orotic acid output. It was concluded that ammonia stimulates the de novo synthesis of pyrimidine in the sheep liver but the participation in this effect of carbamyl phosphate generated in the urea cycle is limited.
Amino Acids↗
[Differences in the cytotoxicity mechanisms of pancreatic and microbial ribonucleases].
The effect of pancreatic and microbial ribonucleases (RNAses) on incorporation of labeled precursors, i. e. 14C-uridine, 14C-thymidine and 14C-glycine into the biopolymers of transplantable cell cultures, as well as their effect on radioactivity of the acid insoluble fraction of the cells labeled with 14C-orotic acid was studied. It was shown that the pancreatic RNAses and RNAses of Streptomyces rimosus in a dose of 1 ED50 determined by the index of the cell monolayer intactness inhibited incorporation of the labeled precursors. The inhibition was rather deep and almost equal. The enzymes lowered also radioactivity of the cells labeled with 14C-orotic acid in proportion to the time of the cell incubation in the presence of the enzymes. On the contrary, RNAses of Bacillus sp. used in analogous doses was significantly less intensive in inhibition of the precursor incorporation and almost did not lower radioactivity of the acid insoluble fraction of the cells totally labeled with 14C-orotic acid.
Animals↗
Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.
Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.
Animals↗