Purification of a RNA-binding protein from rat liver. Identification as ferritin L chain and determination of the RNA/protein binding characteristics

In cultured rat hepatocytes the degradation of phosphoenolpyruvate carboxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability. The 3'-untranslated region of phosphoenolpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA 3'-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphorylation with cAMP-dependent protein kinase and was augmented after dephosphorylation with alkaline phosphatase. Weak binding was observed in extracts from okadaic acid-treated cultured hepatocytes compared with untreated cells. Preincubation of ferritin with an anti-phosphoserine or an anti-phosphothreonine antibody attenuated binding to RNA, while an anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues were in close proximity to the RNA-binding region. Ferritin is the iron storage protein in the liver. Binding of ferritin to RNA was diminished in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect. It is concluded that ferritin might function as RNA-binding protein and that it may have important functions in the general regulation of cellular RNA metabolism.     

Partial isolation and translation in vitro of messenger ribonucleic acid for phosphoenolpyruvate carboxykinase (guanosine triphosphate)

1. mRNA was extracted from the livers of starved rats and incubated in a heterologous cell-free protein-synthesizing system derived from rabbit reticulocytes. The presence of newly synthesized phosphoenolpyruvate carboxykinase (GTP) was detected by immunoprecipitation with a specific antibody to the enzyme and analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The synthesis of the enzyme was dependent on the addition of rat liver RNA, whereas RNA isolated from rat spleen was inactive. If ovalbumin and anti-ovalbumin were used to form the immunoprecipitates, no radioactivity that migrated as phosphoenolpyruvate carboxykinase was detected. 3. The optimal concentrations of magnesium acetate and KCl for phosphoenolpyruvate carboxykinase synthesis were determined. 4. When polyribosomal RNA was separated by sucrose-gradient centrifugation, phosphoenolpyruvate carboxykinase mRNA migrated between 20 and 26 S in keeping with the high mol. wt. of the protein (85 000). 5. The presence of poly(A) in phosphoenolpyruvate carboxykinase mRNA was suggested by retention of mRNA activity on oligo(dT)-cellulose columns. 6. It was concluded that the cell-free synthesis of phosphoenolpyruvate carboxykinase can serve as a bioassay for intracellular phosphoenolpyruvate carboxykinase mRNA.     

Messenger RNA for renal phosphoenolpyruvate carboxykinase (GTP). Its translation in a heterologous cell-free system and its regulation by glucocorticoids and by changes in acid-base balance

A translational assay was used to measure the level of mRNA coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in the rat kidney in various conditions in which the enzyme is induced. RNA extracted from whole kidneys was chromatographed on oligo(dT)-cellulose to select poly(A)-containing RNA. This crude mRNA preparation was able to stimulate amino acid incorporation into protein in a cell-free system containing an extract of wheat germ. Phosphoenolpyruvate carboxykinase could be detected among the polypeptides synthesized and quantitated by immunoprecipitation with a monospecific antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of enzyme synthesized was proportional to the quantity of RNA added. The level of mRNA coding for phosphoenolpyruvate carboxykinase is increased 3-fold 6 h after triamcinolone injection. Translatable enzyme mRNA also increases 3-fold within 6 h of the onset of metabolic acidosis caused by an ammonium chloride load. In both cases, the increase in functional mRNA is commensurate with the stimulation of enzyme synthesis measured in vivo. Glucocorticoid administration and acidosis cause additive increases in the level of translatable phosphoenolpyruvate carboxykinase mRNA. The inductive effect of acidosis is preserved in the absence of the adrenals, hypophysis, thyroid, and parathyroids.     

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin

To utilize hepatocytes for insulin-producing surrogate cells, we devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). The expression of PEPCK is down-regulated by insulin, and up-regulated by cAMP and glucagon. To express insulin in hepatocytes, we constructed an adenoviral insulin expression system. After infection, the hepatocytes secreted immunoreactive insulin (IRI) at an increasing rate. IRI secretion increased over four-fold upon stimulation with 300 microM cAMP and 500 microM of the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This increase was also observed with glucagon and IBMX. Production was augmented two-fold by the addition of wortmannin, phosphatidylinositol (PI)-3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. Addition of exogenous insulin to the culture decreased insulin mRNA expression remarkably on Northern blot. Thus, by using a PEPCK promoter for insulin expression, we were able to up-regulate insulin production from hepatocytes with cAMP and glucagon, and down-regulate with insulin itself.     

Sex differentiation of cerebral centers in mammals

The present paper describes intracellular changes in ribonuclease specific activity during Ca2+-induced sporangium formation in the water mold Achyla bisexualis. The enzymes undergo a decrease in activity prior to crosswall formation followed by an increase in activity during spore cleavage. As spore discharge occurs the RNase activity again decreases. A large percentage of the nuclease activity is associated with a lysosomal-like fraction of the cell, but there is also considerably activity associated with nuclear and microsomal fractions. Addition of cycloheximide or actinomycin D at various times during development prevents further decrease or increase in the enzyme activity. Mixing of cell extracts from different developmental stages provides evidence that inhibitors or activators of the enzyme activity are not responsible for the activity levels evident at the different stages. There is a change in the total levels of presumptive mRNA during Ca2+-induced sporangial formation which appears to be associated with the patterns of RNase activity. Utilizing total cellular RNA and Poly(A)+ RNA with the crude ribonuclease preparations, no substrate specificity could be ascertained.     

Polyamine evaluation in serum and erythrocytes of pediatric patients with neoplasms

A major allergen/antigen, Asp fl, secreted by Aspergillus fumigatus exhibits cytotoxicity towards eukaryotic cell lines. Asp fl inhibited protein synthesis in RAW cells with an IC50 of 4.5 nM and also degraded ribosomal RNA of RAW cells at a similar concentration. Ribosomal inactivation by Asp fl may be the probable mechanism for protein synthesis inhibition. Specific ribonuclease activity of Asp fl was observed to be 100,000 U/mg. Presence of strong RNase activity in Asp fl was further confirmed by agar gels containing yeast RNA. Electrophoretic run on agarose gels showed that Asp fl degrades all species of naked RNA. Modification of histidine residues of Asp fl with diethyl pyrocarbonate and alkylation of cysteines with iodoacetamide resulted in loss of ribonuclease activity and cytotoxicity of Asp fl. The current study establishes the ribonuclease activity of a purified major allergen of A. fumigatus that inhibits protein synthesis and kills the eukaryotic cells.     

Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation

Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described. It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA. Sauromatum but not Olisthodiscus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure. Nuclease levels were monitored by coincubating [3H]-labeled Escherichia coli ribosomal RNA with the experimental RNA samples. The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.     

Characterization of nitric oxide dependent changes in carbohydrate hepatic metabolism during septic shock

The role of nitric oxide in the alterations of liver carbohydrate metabolism during septic shock has been studied in fed and starved animals injected with bacterial lipopolysaccharide (LPS). One h after LPS injection an hyperglycemic peak was observed followed by hypoglycemia when the plasma nitric oxide concentration increased. However, in animals pharmacologically treated with nitric oxide donors only hypoglycemia was observed. In isolated hepatocytes from LPS treated rats an impairment of the gluconeogenic flux was observed accompanied by a decrease in the mRNA levels of the glucose transporter GLUT-2 and phosphoenolpyruvate carboxykinase, at the time that increased the mRNA levels of the inducible form of nitric oxide synthase. These results suggest that part of the effects observed in response to LPS challenge are due to early signaling molecules (cytokines and other factors molecules) whereas other effects can be attributed to nitric oxide synthesis which in turn has specific effects on hepatic metabolism.     

Insulin effects on CYP2E1, 2B, 3A, and 4A expression in primary cultured rat hepatocytes

Although hyperketonemia and/or altered growth hormone secretion caused by diabetes have been implicated in enhanced CYP2E1, 2B, 3A and 4A expression, the effect of insulin on hepatic P450 expression, in the absence of associated metabolic/hormonal alterations, remains unknown. Primary cultured rat hepatocytes have been shown (Zangar et al., Drug Metab. Dispos., 23:681, 1995) to express stable and inducible CYP2E1 mRNA and protein levels, and provide an excellent system for mechanistic examination of the effect of insulin on CYP2E1, 2B, 3A and 4A expression. Maintaining primary rat hepatocytes in culture in the absence of insulin for 48, 72, or 96 h increased CYP2E1 mRNA levels 5-, 11-, and 4-fold, respectively, relative to cells maintained in the presence of the standard concentration of 1 microM insulin. In contrast, CYP2B mRNA levels increased only approximately 2-fold in the absence of insulin, when compared with the presence of 1 microM insulin. CYP2E1 and 2B protein levels were increased 6.7- and 3.8-fold, respectively, in cells cultured for 96 h in the absence of insulin as compared with those cultured in medium containing 1 microM insulin. Concentration-response studies revealed that decreasing the concentration of insulin below 10 nM (i.e. 1 nM, 0.1 nM, no insulin) increased CYP2E1 mRNA levels 4-, 7-, and 11-fold, respectively. In contrast, no such concentration-dependence was observed for CYP2B mRNA expression. As CYP3A and 4A expression is also elevated in diabetic rats, the effects of insulin on these P450s was also examined. CYP3A mRNA levels were unaltered and CYP4A mRNA levels were decreased marginally (approximately 50%) by the absence of insulin relative to levels in cells cultured in the presence of 1 microM insulin over 96 h in culture. The results of this study provide evidence that insulin itself, in the absence of other diabetes-induced metabolic or hormonal alterations, affects CYP2E1 and 2B, but not CYP3A or 4A, expression in primary cultured rat hepatocytes. Furthermore, CYP2E1 expression is differentially regulated by insulin relative to CYP2B, 3A or 4A. This study also demonstrates that decreasing the concentration of insulin in the culture medium provides a method by which CYP2E1 levels can be increased in primary cultured hepatocytes to facilitate mechanistic studies on the regulation of CYP2E1 expression.     

Endotoxin causes reciprocal changes in hepatic nitric oxide synthesis, gluconeogenesis, and flux through phosphoenolpyruvate carboxykinase

Treatment of rats with bacterial endotoxin resulted in a significant induction of hepatic nitric oxide synthase within 3 hours. The response was maximal at 12 hours and was maintained over 18 hours. The induction of nitric oxide synthase correlated well with the increase in plasma nitrate plus nitrite concentrations and also with the inhibition of glucose synthesis in subsequently isolated hepatocytes. The decline in the rate of gluconeogenesis also correlated with an inhibition of flux through phosphoenolpyruvate carboxykinase but not with alterations in flux through either pyruvate kinase or 6-phosphofructo-1-kinase, suggesting that a nitric oxide-induced inhibition of phosphoenolpyruvate carboxykinase may underlie the decreased glucose production in sepsis.     

Spermidine-induced destabilization of ornithine decarboxylase (ODC) is mediated by accumulation of antizyme in ODC-overproducing variant cells

The mechanism of spermidine-induced destabilization of ornithine decarboxylase (ODC) was examined in newly isolated ODC-overproducing variant cells by use of an in vitro ODC degrading system. The cells accumulated ODC protein in the presence of alpha-difluoromethylornithine. Addition of spermidine to the medium accelerated degradation of ODC protein concomitantly with induction of antizyme, a regulatory protein that binds to ODC, inhibiting its activity. Both the acceleration of ODC degradation and the induction of antizyme were inhibited by cycloheximide, but not by actinomycin D. ODC was degraded rapidly in extracts from spermidine-treated cells. The rate of ODC degradation correlated with the amount of antizyme in the extracts, and the degradation activity was abolished by treatment of the extracts with anti-antizyme antibody. Thus, antizyme induced by spermidine was essential for the accelerated degradation of ODC in the cells. ODC was phosphorylated in the cells, probably at serine residue 303 in the first internal PEST region. ODC phosphorylation occurred even when its new synthesis was inhibited by cycloheximide. Antizyme accelerated the degradations of both dephosphorylated ODC and native ODC.     

Regulation of phosphoenolpyruvate carboxykinase and fatty acid synthetase in brown fat of suckling rats

Infant rats were injected with prednisolone (0.5-5 mg/100 g wt). This caused phosphoenolpyruvate carboxykinase (PEPCK) activity to rise in liver and to decrease in brown fat. Fatty acid synthetase (FAS) activity remained unchanged in liver but increased in brown fat. A single injection of prednisolone caused hepatic PEPCK activity to remain elevated for at least 7 days. Brown fat FAS also remained high for that period. However, brown fat PEPK activity returned to normal on the third day after the injection. A single injection of prednisolone or cortisone to 5-day-old rats caused a transient elevation of the blood level of insulin and a prolonged decrease in that of growth hormone. No effect on the level of glucagon was noted. Injections of insulin had effects similar to those of prednisolone, i.e. a rise in hepatic and a fall in brown fat PEPCK. Using antibodies prepared to hepatic PEPCK it was shown that the observed changes were due to changes in the rate of synthesis of the enzyme. Using actinomycin D indirect evidence was obtained that changes in FAS activity of brown fat were also due to changes in the synthetic rate.     

Biomonitoring using accessible human cells for exposure and health risk assessment

We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding beta-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold enhanced by silver and gold toning, and fluorescently labeled tyramides. Diaminobenzidine and Nanogold can be used in both light and electron microscopy. The nickel-enhanced diaminobenzidine was the most sensitive visualization method. It is easy to accomplish but a drawback is poor spatial resolution, which restricts its use at high magnifications. Nanogold visualization has considerably better spatial resolution and is therefore recommended for electron microscopy. Fluorescent tyramides, especially TRITC-tyramide, offer a good detection method for fluorescence and confocal microscopy. The methods were used to localize amphoterin and beta-actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, beta-actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, beta-actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen. (J Histochem Cytochem 47:99-112, 1999)     

Neurological monitoring of neurotoxicity induced by paclitaxel/cisplatin chemotherapy

Thiazolidinediones are potent antidiabetic compounds, in both animal and human models, which act by enhancing peripheral sensitivity to insulin. Thiazolidinediones are high-affinity ligands for peroxisome proliferator-activated receptor-gamma, a key factor for adipocyte differentiation, and they are efficient promoters of adipocyte differentiation in vitro. Thus, it could be questioned whether a thiazolidinedione therapy aimed at improving insulin sensitivity would promote the recruitment of new adipocytes in vivo. To address this problem, we have studied the in vivo effect of pioglitazone on glucose metabolism and gene expression in the adipose tissue of an animal model of obesity with insulin resistance, the obese Zucker (fa/fa) rat. Pioglitazone markedly improves insulin action in the obese Zucker (fa/fa) rat, but doubles its weight gain after 4 weeks of treatment. The drug induces a large increase of glucose utilization in adipose tissue, where it stimulates the expression of genes involved in lipid metabolism such as the insulin-responsive GLUT, fatty acid synthase, and phosphoenolpyruvate carboxykinase genes, but decreases the expression of the ob gene. These changes are related to both an enhanced adipocyte differentiation, as shown by the large increase in the number of small adipocytes in the retroperitoneal fat pad, and a direct effect of pioglitazone on specific gene expression (phosphoenolpyruvate carboxykinase and ob genes) in mature adipocytes.     

Globin messenger RNA from anaemic rabbit spleen. Size of its polyadenylate segment

The size of the polyadenylate segment of globin messenger RNA isolated from spleens of anaemic rabbits was estimated by comparison of its electrophoretic migration in polyacrylamide gels to that of synthetic poly(A) segments of known lengths. Conditions of enzymic degradation of mRNA with pancreatic ribonuclease and T1 ribonuclease were carefully established in order to ensure complete degradation of the heteropolymeric part of mRNA without affecting the polyadenylate sequence. The poly (A) segments of spleen globin mRNA were found to be 25-90 nucleotides long whilst those of peripheral blood reticulocytes from the same animals were only 10-30 residues long. Since spleen contains young erythroid cells and since anucleated blood reticulocytes constitute a statistically older population of the same cell line, these results support the idea that the poly(A) segment of mRNA shortens when the message ages.