Endoplasmic reticulum nuclease. Purification and specificity

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver endoplasmic reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 X 10(4), 9 X 10(4), 1.55 X 10(5) and Sephacryl S-200 fraction at V0). Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis (Mr 5.4 X 10(4). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphates can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly Mg2+. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and DNA . RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.     

Vitamin B6-deficient diet plus 4-deoxypyridoxine (4-DPD) reduces the inflammatory response induced by T. spiralis in diaphragm,masseter and heart muscle tissue of mice

Animals fed diets deficient in vitamin B6 develop microcytic anemia, alterations of growth, and other pathologies. 4-deoxypirydoxine is a potent antagonist of vitamin B6 coenzyme which depresses IL-1, TNF and IL-6 and has anti-inflammatory properties. The aim of this study was to show the anti-infl ammatory effects of 4-DPD on chronic inflammation caused by the nematode parasite T. spiralis, specifically on the recruitment and the activation of inflammatory cells. Two groups of mice, 6 weeks of age, were used: one was maintained on a vitamin B6-deficient synthetic pellet diet for 15 days before injection of the nematode, and administered an intraperitoneal injection (i.p.) of 4-DPD (250 g/mouse) for 15 days (the first, 5 days before infection), and the second group was maintained on a normal diet for the total duration of the experiment. These two groups were then injected with 150 larvae (L1-T. spiralis) per os.Chronic inflammation was caused by infection of treated or untreated mice with T. spiralis parasite. After 14 days post-infection all mice developed a chronic inflammatory response. Mice fed with a B6-deficient diet showed a significant decrease in the number of cysts found in the diaphragm when compared to mice treated with normal diet. In addition, in all mice treated with vitamin B6-deficient diet plus 4-DPD the average body weight was significantly lower, compared to the mice on normal diet in all weeks examined. Moreover, in sections of the diaphragm, masseter and miocardium muscles, the infiltration of inflammatory cells, such as macrophages, lymphocytes, and eosinophils were more intense in untreated mice compared to those fed a vitamin B6-deficient diet.These results show that BALB/c mice infected with T. spiralis and fed a vitamin B6-deficient diet plus the vitamin B6 antagonist, 4-DPD, prolong the time of invasion of the larvae in the muscle cells, influence the recruitment of inflammatory cells and the intensity of the inflammatory reaction compared to infected untreated mice (control)     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Induction of monocyte chemotactic protein-1 (MCP-1) and TNF alpha by Trichinella spiralis in serum of mice in vivo

Effects of selenium deficiency, induced by thioacetamide, were investigated in rats. Thioacetamide (0.3 g/L) given in drinking water, as expected, caused a significant loss of selenium from the liver. It was accompanied by liver cirrhosis and a significant increase in the liver weight as well as liver to body weight ratio. A significant loss of selenium from spleen was also accompanied by an increase in its weight. Weights of lungs, testis and kidney, however, were not affected by thioacetamide and there was no change in their selenium content. Plasma levels of selenium were significantly reduced in the thioacetamide treated group. All these changes were confirmed to be due to selenium deficiency caused by thioacetamide, as supplementation with selenium reversed these changes. The mode of action of selenium is unknown but may involve anti-oxidant defense mechanisms.     

4-Deoxypyridoxine inhibits chronic granuloma formation induced by potassium permanganatein vivo

4-Deoxypyridoxine (4-DPD) is a potent antagonist of Vitamin B6 coenzyme which inhibits IL-1, lymphocyte proliferation and has demonstrated that tollerance to skin grafts can be induced by administering splenic cells to pyridoxine-deficient mice. Chronic inflammation induced by dorsal injections of 200 l of a 140 saturated crystal solution of potassium permanganate (KMnO₄) in mice treated or untreated with 4-DPD (400 g/dose), has been investigated. After 7 days all mice developed a subcutaneous granulomatous tissue indicative of a chronic inflammatory response, at the site of injection. KMnO₄-treated mice injected intraperitoneally with 4-DPD (400 g/dose) on 5 consecutive days (the first at the same time of induction of the granuloma) show a significant decrease in size and weight of granuloma when compared to mice not treated with 4-DPD (Controls). In addition, in all mice treated with 4-DPD there was a strong inhibition of TNF in serum (P<0.01) and in supernatant fluids (P<0.05) from minced granuloma, while IL-6 was inhibited in the supernatant fluids (P<0.05) of minced granulomas but was not detected in the serum of treated and untreated mice. In this study we show for the first time the antiinflammatory effect of 4-DPD on chronic inflammation and the inhibitory effect of TNF and IL-6 generation in supernatant fluids from minced granulomas.     

The DNase activity of an endoplasmic reticulum nuclease and its effect on DNA synthesis in vitro

An endoplasmic reticulum nuclease which was isolated previously in this laboratory from rat liver ( Kouidou et al. (1981) Eur.J. Bioch . 120, 9-14) was found to degrade linear and circular single stranded DNA but not double stranded DNA. The DNA fragments resulting from this cleavage were longer than 20 nucleotides. In addition the nuclease was found to improve the efficiency of DNA template used by DNA polymerase I in DNA synthesis in vitro. The results were the same whether incubation of the template with the nuclease was prior to addition of DNA polymerase I or simultaneously with polymerization. When nuclease was added after the completion of polymerization by DNA polymerase I it was ineffective unless the product was denatured. These data further corroborate the observation that double stranded DNA is not cleaved by this enzyme.     

DNA-dependent RNA polymerase from T8 Guerin tumor

DNA-dependent RNA polymerases from nuclei of T8 Guerin tumor were studied. Two enzymes were purified several hundred times by the use of ammonium sulfate precipitation, DEAE-cellulose and phosphocellulose chromatography. One of them belongs to A(I) RNA polymerases and the second to B(II) as was established from their metal and ionic strength requirements. activity in the presence of native and denatured DNA and the resistance to a-amanitin inhibition. The quantity of class A enzyme was increased compared to B, a fact observed with most neoplastic tissues so far studied. This increase of the polymerase responsible for ribosomal RNA synthesis could probably be related to malignant transformation in animals.     

Single-strand-specific nuclease from rat liver endoplasmic reticulum: characterization and mode of action

1. The characteristics and mode of action of a single-strand-specific nuclease isolated from rat liver endoplasmic reticulum are investigated with respect to its DNA and RNA substrates. 2. The RNase activity of the enzyme is slightly influenced by the presence of divalent cations but the DNase activity is enhanced by divalent cations particularly Mn2+. 3. Activity is partially inhibited by the presence of EGTA; this effect is reversed most efficiently by the addition of Mn2+. 4. The enzyme exhibits small pH dependence between pH 6-9 and maximum activity is observed at pH 7-7.5 for both DNase and RNase activities. 5. Sulfhydryl group reagents do not affect its action but histidyl group reagents exert a small but definite effect. 6. The enzyme degrades DNA and RNA endonucleolytically producing fragments which possess 3'-OH and 5'-phosphate termini. 7. Monomers are not produced even after prolonged degradation. 8. The end product of poly(U)degradation ranges between two and four building blocks but the DNA product is longer probably due to considerable percentage of secondary structure.