Studies on liver mitochondrial 5′-endonuclease activity in rats fed carcinogenic amines and on their binding to mitochondrial proteins

Mitochondrial 5′-endonuclease activity has been determined at regular time intervals in the livers of rats fed a diet containing 0.09% 2-aminofluorene (AF), 0.09% 2-acetylaminofluorene (AAF) or 0.06% N,N-dimethyl-4-aminoazobenzene (DAB). The results obtained indicate that the 5'-endonuclease activity was not affected significantly.The quantity of AF, AAF or DAB bound to liver homogenate and mitochondrial fraction proteins has also been measured at regular time intervals. The amount of AF and AAF bound to homogenate proteins after 4 weeks of carcinogen feeding is about 60-fold higher than that of DAB. The binding of the AF compounds to the mitochondrial fraction proteins is comparatively more important, reaching a level 300-fold higher than that of DAB. The amount of AF residues bound per mg of mitochondrial fraction proteins is higher than that of the homogenate while that of rats fed DAB is smaller. The present results suggest that no relation can be established between the total amount of these carcinogens bound to liver cellular proteins in vivo and their potential carcinogenic effect.     

The identification of the site of action of NN′-dicyclohexylcarbodi-imide as a proteolipid in mitochondrial membranes

Mitochondrial membranes were incubated with NN'-dicyclohexyl[(14)C]carbodi-imide, which irreversibly inhibited the partial reactions of oxidative phosphorylation by 95-100%. Solutions of the membranes were analysed on polyacrylamide gels. Of the radioactivity recovered from the gels 90% was shown to be associated with a single protein of molecular weight about 10000. The radioactive protein and associated phospholipid was solubilized from the membrane by extraction with chloroform-methanol mixtures and was concentrated 50-fold by solvent fractionation and adsorption chromatography on Sephadex LH-20. Several protein-radioactivity peaks were obtained by Sephadex LH-20 chromatography. However, 90-100% of the radioactivity in each peak was shown to be associated with a single protein similar to the major radioactive protein observed in electrophoretograms of the membrane solutions. It is concluded that dicyclohexylcarbodi-imide inhibits mitochondrial oxidative phosphorylation by reacting covalently with a group on this chloroform-methanol-soluble protein. The possible role of this protein in oxidative phosphorylation is discussed.     

α2 and β-adrenergic stimulation of corticosterone secretion in rats

Bilateral injection of 6-hydroxydopamine into the medial forebrain bundle (MFB) significantly decreased monoamine concentrations in the hypothalamus. The noradrenaline and serotonin content of the paraventricular nucleus (PVN) was also significantly reduced. These drastic decreases in neurotransmitter concentration did not alter basal secretion of corticosterone. Isoproterenol. a -adrenoceptor agonist (1 mg/kg, i.p.), significantly stimulated corticosterone release in saline and MFB lesioned rats. This stimulation did not differ significantly between the two groups. Clonidine, an ₂-adreceptor agonist, injected either intraperitoneally or intracerebrally just dorsal to the PVN, caused a dose-dependent increase in corticosterone secretion. The stimulation of corticosterone release by clonidine (250 g/kg, i.p.) was antagonised by the selective ₂-adreceptors antagonist, yohimbine (1 mg/kg, i.p.) and significantly reduced by the MFB lesion. These results suggest that corticosterone secretion is stimulated by activation of ₂-adreceptors which occur on noradrenergic nerve terminals in the PVN.     

No role of the 5′ untranslated region of ornithine decarboxylase mRNA in the feedback control of the enzyme

The polyamines are ubiquitous in nature and appear to fulfil several important functions, mostly related to growth, in the cell. The first, and often rate-limiting, step in the biosynthesis of the polyamines is catalysed by ornithine decarboxylase (ODC), which is subject to a variety of control mechanisms. The polyamines exert a strong feedback regulation of the expression - as well as the degradation of the enzyme. The regulation of ODC expression appears to occur at the translational level. The ODC mRNA contains a long GC-rich 5 untranslated region (UTR), which has been demonstrated to hamper the translation of the mRNA. However, it has not yet been conclusively established whether this part of the mRNA fulfils any function in relation to the polyamine-mediated control of ODC synthesis. In the present study, we have used stable transgenic CHO cells, expressing either full-length ODC mRNA or 5 UTR-truncated ODC mRNA, to elucidate the role, if any, of the 5 UTR in the translational regulation of the enzyme by polyamines. No differences in regulatory properties were observed between the cells expressing the full-length ODC mRNA and those expressing the ODC mRNA devoid of most the 5 UTR. The cell lines down-regulated ODC (synthesis as well as activity) to the same extent upon exposure to an excess of polyamines, demonstrating that the feedback control of ODC mRNA translation occurs by a mechanism independent of the major part of the 5 UTR of the ODC mRNA.     

Selective stimulation by tri-iodothyronine of myocardial deoxyribonucleic acid polymerase-α in neonatal rats

Three forms of DNA polymerase (alpha, beta and gamma) were separated from isolated rat myocardial cells on the basis of template, pH and ionic requirements, sensitivity to N-ethylmaleimide and position on sucrose gradients. Tri-iodothyronine administration (20mug/100g intraperitoneally) to 3-week-old rats resulted in selective stimulation of DNA polymerase-alpha (198+/-7.1 versus 102+/-5.8pmol of [(3)H]dTMP/30min per mg of protein in untreated controls, P<0.01), with no change in polymerases-beta and -gamma. [(3)H]Thymidine incorporation into myocardial DNA was also enhanced in tri-iodothyronine-treated neonatal rats (132+/-11.2 versus 53+/-4.1c.p.m./mug of DNA in controls, P<0.001). Increased incorporation was associated with an expansion of deoxyribonucleoside 5'-triphosphate pools, especially that of dTTP (24+/-1.6 versus 10+/-1.1pmol/mg of DNA, P<0.01). Neither DNA polymerase activities nor [(3)H]thymidine incorporation were changed in 6-month-old rats in response to tri-iodothyronine. Unstimulated adult myocardial cells had DNA polymerase activities comparable with those in 3-week-old animals, but significantly lower [(3)H]-thymidine incorporation and deoxyribonucleoside triphosphate concentrations. Enhancement of both DNA polymerase-alpha activity and [(3)H]thymidine incorporation in tri-iodothyronine-treated young rats was prevented by concomitant administration of either vinblastine (1mug/g) or daunomycin (2mug/g); actinomycin D (0.1mug/g) or cycloheximide (8mug/g), on the other hand, prevented the increase in [(3)H]thymidine incorporation, but not DNA polymerase-alpha activation. These results demonstrate an age-dependent stimulation of myocardial DNA replication by tri-iodothyronine and suggest an inter-relationship between DNA synthesis and subsequent entry into mitosis.     

Effect of selenite and selenate on rat liver nuclear 3,5,3′-triiodothyronine (T3) receptor

The present study was undertaken to investigate the effects of selenite (Se^IV) or selenate (Se^VI) on nuclear T₃ receptors of rat liver. Selenite at 0.1 μM (p<0.01) inhibited the T₃ specific binding to rat liver nuclear receptors. The specific binding of the T₃ receptor was fully restored when even 1.0 μM selenite was separated from the T₃ receptor by gel filtration. No inhibitory effect of selenite (up to 100 μM) on the T₃ binding to nuclear receptor was found in the presence of 1.0 mM dithiothreitol. The rate of dissociation of the T₃-nuclear receptor complex was effectively increased by 0.1 μM selenite. Selenate up to 1 mM as well as sulfite or sulfate up to 0.1 mM did not exert an inhibitory effect on T₃ receptors. The results based on the in vitro experiments suggest that the selenium in the form of selenite may reversibly affect the T₃ binding on the receptor molecule.     

Study of the therapeutic effects of Lactobacillus and α-lipoic acid against dimethylnitrosamine-induced liver fibrosis in rats

Lactobacillus (LB) and α-lipoic acid (ALA) were investigated to compare their protective effects against dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Animals were either injected intraperitoneally with DMN to induce hepatic fibrosis, or were left untreated (negative control). For the DMN + LB and DMN + ALA treatment groups, at two weeks of DMN treatment LB or ALA was added to the feed and supplementation continued until the experimental endpoint at sixty days. At the study endpoint, expression of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, TGF-β1, COL1-α1 genes and the concentration of glutathione and malondialdehyde were measured in liver tissues, while GOT, GPT, and ALP concentrations were measured in blood. Body weights remained higher in NC and DMN + LB groups compared to DMN and DMN + ALA groups, while activity of GOT and GPT in serum was lower in DMN + LB and DMN + ALA groups compared to the DMN group. Compared to other treatment groups, in the DMN group expression of both TGF-β1 and, COL1-α1 mRNAs and pro-inflammatory cytokines increased, while that of 1L-10 decreased. Furthermore, LB and ALA treatments increased antioxidant activity of glutathione and decreased malondialdehyde in comparison to the DMN group. Between LB and ALA treatments, glutathione concentration was higher in the DMN + LB group, while malondialdehyde was lower. Our results indicate that both LB and ALA exert hepatoprotective effects against DMN-induced liver fibrosis. Their beneficial effects may be partly associated with down-regulation of both TGF-β1 and COL1-α1 signaling, which may be accounted for reduction of increased oxidative stress and TNF-α production.     

Compartmentation of uridine 5′-triphosphate occurs during synthesis of cytoplasmic and mitochondrial ribosomal RNAs of cultured tomato cells but not during synthesis of cytoplasmic ribosomal and transfer RNAs

Compartmentation of uridine 5-triphosphate (UTP) was studied during the nucleolar synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and the synthesis of cytoplasmic transfer RNA (cyt-tRNA) in the nuclear matrix as well as the synthesis of mitochondrial ribosomal RNA (mt-rRNA) in tomato (Lycopersicon esculentum Mill. cv. Lukullus) cell-suspension culture using the approach of Wiegers et al. (Eur. J. Biochem. 64, 535–540, 1976). Before measurements were made, it was ensured that: (i) there was steady-state labeling of all RNAs studied as well as UTP; (ii) there was stability of cyt-tRNA and cyt-rRNA; (iii) there was no label randomization through degradation of [³H]uridine; (iv) there were significant differences in the specific radioactivity of UTP, the final immediate precursor of RNA, after supplying the cells with two different exogenous [³H]uridine concentrations.By comparing the steady-state specific radioactivity of UTP with that of cyt-tRNA and cyt-18S rRNA during constant [³H]uridine supply, we found that the three molecules had equal specific radioactivities which, however, differed significantly from that of the mt-rRNA. With a 20-fold higher uridine concentration, i.e. a 20-fold lower specific radioactivity of exogenous [³H]uridine, the specific radioactivity of cyt-rRNA, cyt-tRNA and UTP decreased proportionally whereas that of mt-RNA increased. These results argue against different UTP pools during synthesis of cyt-rRNA and cyt-tRNA, but indicate compartmentation of UTP during rRNA synthesis in the nucleus and the mitochondria of tomato cells.Abbreviations CMP cytidine 5-monophosphate - cyt-rRNA cytoplasmic ribosomal RNA - cyt-tRNA cytoplasmic transfer RNA - mt-rRNA mitochondrial rRNA - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - TLC thin-layer chromatography - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP uridine 5-diphosphate - UMP uridine 5-monophosphate - UTP uridine 5-triphosphate     

Transport and distribution of α-tocopherol in lymph,serum and liver cells in rats

Rats were cannulated in the major mesenteric lymph duct and given an intraduodenal bolus of unlabeled and α-[³H]tocopherol, and [¹⁴C]oleic acid in soybean oil. The appearance of α-tocopherol in lymph was negligible during the first 2 h and peaked 4–15 h after feeding, whereas no detectable amount was recovered in the portal vein. Intestinal absorption via the lymphatic pathway was 15.4 ± 8.9% (n = 10) and 45.9 ± 10.8% (n = 4) for α-tocopherol and [¹⁴C]oleic acid, respectively. About 99% of α-tocopherol in lymph was associated with the chylomicron fraction (d < 1.006 g/ml). In non-fasting rats, 51% of serum α-tocopherol was associated with chylomicrons/VLDL (very-low-density lipoprotein, d < 1.006 g/ml) and 47% with HDL (high-density lipoprotein, 1.05 < d < 1.21 g/ml). Our study revealed that the liver, skeletal muscle and adipose tissue contain approx. 92% of the total mass of α-tocopherol measured in ten different organs. Parenchymal and nonparenchymal liver cells contributed to 75% and 25% of the total mass of α-tocopherol in the liver, respectively.     

Ferrochelatase and δ-aminolaevulate synthetase in brain, heart, kidney and liver of normal and porphyric rats. The induction of δ-aminolaevulate synthetase in kidney cytosol and mitochondria by allylisopropylacetamide

1. delta-Aminolaevulate synthetase was detected in liver and kidney mitochondria prepared from normal rats. 2. The administration of allylisopropylacetamide induced an increase in delta-aminolaevulate synthetase in both liver and kidney mitochondria and the enzyme also appeared in the cytosol fraction of both tissues. Comparison with the distribution of glutamate dehydrogenase indicated that this soluble kidney delta-aminolaevulate synthetase was truly of cytosol origin and did not arise from disrupted mitochondria. The kidney cytosol enzyme was inhibited by 50% by 50mum-protohaem. 3. delta-Aminolaevulate synthetase could not be detected in mitochondria or cytosol from heart or brain from normal or porphyric rats. 4. The administration of allylisopropylacetamide caused little or no increase in ferrochelatase or cytochrome content of liver, kidney, heart or brain mitochondria.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

The 3′ → 5′ exonucleases of both DNA polymerases δ and ε participate in correcting errors of DNA replication in Saccharomyces cerevisiae

DNA polymerases II (ε) and III(δ) are the only nuclear DNA polymerases known to possess an intrinsic 3′ → 5′ exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase δ and ε 3′ → 5′ exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10² and 10¹, respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 × 10³-fold. Analysis of mutation rates of double mutants indicated that the 3′ → 5′ exonucleases of DNA polymerases δ and ε can act competitively and that, like the 3′ → 5′ exonuclease of DNA polymerase δ the 3′ → 5′ exonuclease of DNA polymerase ε acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3′ → 5′ exonucleases of DNA polymerases δ and ε act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.     

The effect of the use of copper carbonate and copper nanoparticles in the diet of rats on the level of β-amyloid and acetylcholinesterase in selected organs

BackgroundCopper has an important role in nervous system function, as a cofactor of many enzymes and in the synthesis of neurotransmitters. Both the dose and the chemical form of copper can determine the impact of this element on metabolism, the neurological system and the immune system.AimsThe aim of the study was to determine whether and in what form the addition of copper changes the level of amyloid beta and acetylcholinesterase level in selected rat tissues.MethodsThirty, healthy, male, albino Wistar rats aged 7 weeks were randomly divided into 3 groups. Three experimental treatments were used to evaluate the effects of different levels and sources of Cu (6.5 mg kg of diet) in the diet: Cu₀ – rats fed a diet without Cu supplementation; Cu_salt – rats fed a diet with CuCO₃ (6.5 mg kg of diet) during two months of feeding; Cu_NPs - rats fed a diet with Cu nanoparticles (6.5 mg kg of diet) during two months of feeding. In blood serum and tissue homogenates there rated the indicators proving the potential neurodegenerative effect and epigenetic DNA damage induced by chemical form of copper or lack of additional copper supplementation in diet were determined. There were analysed: level of acetylcholinesterase, β-amyloid, low-density lipoprotein receptor-related protein 1, apyrimidinic endonuclease, thymidine glycosidase, alkylpurine-DNA-N-glycosylase and glycosylated acetylcholinesterase.ResultsIrrespective of the form of copper added, it was found to increase acetylcholinesterase level in the brain, spleen and liver, as well as in the blood plasma of the rats. Copper in the form of CuCO₃ was found to increase acetylcholinesterase level in the kidneys. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu.ConclusionsA lack of added Cu in the diet of rats reduces the concentration of amyloid-β in the blood, whereas administration of copper, in the form of either CuNPs or CuCO₃, increases the level of this peptide in the blood. The use of copper in the form of CuNPs in the diet of rats does not increase the level of β-amyloid more than the use of the carbonate form of this element. The use of CuNPs or CuCO₃ in the diet of rats increases acetylcholinesterase level in the brain, spleen, liver, and blood. CuNPs in the diet of rats were not found to increase acetylcholinesterase level to a greater extent than Cu⁺² carbonate.     

The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation

The degradation of eukaryotic mRNAs can be initiated by deadenylation, decapping, or endonuclease cleavage. This is followed by 5′–3′ degradation by homologs of Xrn1, and/or 3′–5′ degradation by the exosome. We previously reported that, in African trypanosome Trypanosoma brucei, most mRNAs are deadenylated prior to degradation, and that depletion of the major 5′–3′ exoribonuclease XRNA preferentially stabilizes unstable mRNAs. We now show that depletion of either CAF1 or CNOT10, two components of the principal deadenylation complex, strongly inhibits degradation of most mRNAs. RNAi targeting another deadenylase, PAN2, or RRP45, a core component of the exosome, preferentially stabilized mRNAs with intermediate half-lives. RRP45 depletion resulted in a 5′ bias of mRNA sequences, suggesting action by a distributive 3′–5′ exoribonuclease. Results suggested that the exosome is involved in the processing of trypanosome snoRNAs. There was no correlation between effects on half-lives and on mRNA abundance.     

The chirality breaking in the photolysis of leucine enantiomers and 5′CMP

The inhibition of the photohydrate formation of the cytidine 5 monophosphate (5 CMP) under UV irradiation in the presence of the D-, L-leucine in phosphate aqueous solution (pH 7) has been studied. L-leucine demonstrates more effective in preventing 5 CMP from photohydration than that of D-leucine. This photolysis method can be used instead of NMR spectroscopy to research weak nucleotide-amino acid interaction in aqueous solution especially for nonaromatic acid. A statistical thermodynamic stacking model is proposed to describe the mechanism producing chiral symmetry breaking. The calculated weak interaction energy is 1.16kT and 2.05kT for D-, and L-leucine respectively.