Nucleic Acid Degradation Products Research Articles

Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells

BackgroundTo better assess potential hepatotoxicity of nanomaterials, human liver HepG2 cells were exposed for 3 days to five different CeO2 (either 30 or 100 μg/ml), 3 SiO2 based (30 μg/ml) or 1 CuO (3 μg/ml) nanomaterials with dry primary particle sizes ranging from 15 to 213 nm. Metabolomic assessment of exposed cells was then performed using four mass spectroscopy dependent platforms (LC and GC), finding 344 biochemicals.ResultsFour CeO2, 1 SiO2 and 1 CuO nanomaterials increased hepatocyte concentrations of many lipids, particularly free fatty acids and monoacylglycerols but only CuO elevated lysolipids and sphingolipids. In respect to structure-activity, we now know that five out of six tested CeO2, and both SiO2 and CuO, but zero out of four TiO2 nanomaterials have caused this elevated lipids effect in HepG2 cells. Observed decreases in UDP-glucuronate (by CeO2) and S-adenosylmethionine (by CeO2 and CuO) and increased S-adenosylhomocysteine (by CuO and some CeO2) suggest that a nanomaterial exposure increases transmethylation reactions and depletes hepatic methylation and glucuronidation capacity. Our metabolomics data suggests increased free radical attack on nucleotides. There was a clear pattern of nanomaterial-induced decreased nucleotide concentrations coupled with increased concentrations of nucleic acid degradation products. Purine and pyrimidine alterations included concentration increases for hypoxanthine, xanthine, allantoin, urate, inosine, adenosine 3′,5′-diphosphate, cytidine and thymidine while decreases were seen for uridine 5′-diphosphate, UDP-glucuronate, uridine 5′-monophosphate, adenosine 5′-diphosphate, adenosine 5′-monophophate, cytidine 5′-monophosphate and cytidine 3′-monophosphate. Observed depletions of both 6-phosphogluconate, NADPH and NADH (all by CeO2) suggest that the HepG2 cells may be deficient in reducing equivalents and thus in a state of oxidative stress.ConclusionsMetal oxide nanomaterial exposure may compromise the methylation, glucuronidation and reduced glutathione conjugation systems; thus Phase II conjugational capacity of hepatocytes may be decreased. This metabolomics study of the effects of nine different nanomaterials has not only confirmed some observations of the prior 2014 study (lipid elevations caused by one CeO2 nanomaterial) but also found some entirely new effects (both SiO2 and CuO nanomaterials also increased the concentrations of several lipid classes, nanomaterial induced decreases in S-adenosylmethionine, UDP-glucuronate, dipeptides, 6-phosphogluconate, NADPH and NADH).

Getting CAD in Shape: The Atomic Structure of Human Dihydroorotase Domain

CAD is a large multifunctional polypeptide that initiates and controls the de novo biosynthesis of pyrimidines in animals. In this issue of Structure, Grande-García and colleagues provide the first atomic information of this antitumoral target by reporting the crystal structure of the dihydroorotase domain of human CAD.

Contribution of the Bacterial Endosymbiont to the Biosynthesis of Pyrimidine Nucleotides in the Deep-sea Tube Worm Riftia pachyptila

The deep-sea tube worm Riftia pachyptila (Vestimentifera) from hydrothermal vents lives in an intimate symbiosis with a sulfur-oxidizing bacterium. That involves specific interactions and obligatory metabolic exchanges between the two organisms. In this work, we analyzed the contribution of the two partners to the biosynthesis of pyrimidine nucleotides through both the "de novo" and "salvage" pathways. The first three enzymes of the de novo pathway, carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, were present only in the trophosome, the symbiont-containing tissue. The study of these enzymes in terms of their catalytic and regulatory properties in both the trophosome and the isolated symbiotic bacteria provided a clear indication of the microbial origin of these enzymes. In contrast, the succeeding enzymes of this de novo pathway, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, were present in all body parts of the worm. This finding indicates that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines. In addition, it suggests that the synthesis of pyrimidines in other tissues is possible from the intermediary metabolites provided by the trophosomal tissue and from nucleic acid degradation products since the enzymes of the salvage pathway appear to be present in all tissues of the worm. Analysis of these salvage pathway enzymes in the trophosome strongly suggested that these enzymes belong to the worm. In accordance with this conclusion, none of these enzyme activities was found in the isolated bacteria. The enzymes involved in the production of the precursors of carbamyl phosphate and nitrogen assimilation, glutamine synthetase and nitrate reductase, were also investigated, and it appears that these two enzymes are present in the bacteria.

Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids.

We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberated from glycoproteins and glycolipids following methanolysis. The stable derivatives of the most common monosaccharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproducibly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound to Asn in N-glycans is quantitatively recovered as two peaks. The latter were easily distinguished from the other GlcNAc residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of soluble or membrane-bound glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degradation products of nucleic acids) do not interfere with these determinations. A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significant interferences. Since fatty acid methyl esters and sphingosine derivatives are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 microg of the initial compound purified by preparative Silicagel TLC. Using electron impact ionization mass spectrometry, reporter ions for the different classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, and KDN) allow the identification of these compounds in very complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive or negative ions. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O-methyl glycosides is achieved.

Changes in the Serum of Mice Infected with Streptococcus Pneumoniae that Stimulate In-vitro Multiplication of Virulent but not Avirulent Strains

During pneumococcal infection of mice, nucleic-acid products, including deoxynucleotides, may be released into the serum from cellular disintegration in at least three organs, the lungs, spleen and liver. The serum, after sterilisation to remove contaminating pneumococci, stimulated multiplication of virulent but not avirulent pneumococci in vitro. It also stimulated growth of virulent pneumococci in serum from uninfected animals and could be replaced, at least in part, by certain nucleic-acid degradation products at concentrations found in infected serum. The effects of the serum were lost after dialysis or dilution.

A comparison of the characteristics of soluble organic nitrogen in untreated and activated sludge treated wastewaters

Soluble organic materials containing nitrogen (SON) are present in effluents from activated sludge treatment of domestic wastewater, but little is known about the sources and characteristics of these materials. The objective of this research was to evaluate the characteristics of SON in untreated wastewaters and activated sludge effluents. Characterization techniques used included low microbial seed biodegradability, molecular weight distribution using gel filtration chromatography, removal by activated carbon and ion exchange, and analysis for free and combined amino acids. Activated sludge effluent SON was more refractory (40–50%; first-order decay rates for the remainder were about 0.014 day −1 than SON in untreated wastewater (18–38%; decay rates for the remainder were 0.08–0.16 day −1). SON produced biologically during treatment had decay rates (about 0.028 day −1) similar to SON in municipal activated sludge effluents, and was from 20 to 100% refractory. Less than 10% of the SON in municipal activated sludge effluent consisted of free or combined amino acids. Approximately 15–30% appeared to nucleic acid degradation products. Fifty to 60% of the SON and SCOD had apparent molecular weights of less than 1800. Apparent molecular weight distributions of treated and untreated wastewaters were similar; however, the excess SON produced during activated sludge start-up contained considerably more SON with molecular weights greater than 1800. The 165–340 molecular weight fraction had a significantly higher SON to SCOD ratio than any other fraction for all wastewaters examined. Activated carbon adsorption efficiently removed SON (72 ± 9%) and SCOD (78 ± 6%) from treated and untreated wastewaters, and from biologically produced organics. Significantly more SON was removed by cationic exchange at pH 2.0 (33–56%) than by anionic exchange at pH 9.5 (10–24%) for all wastewaters tested. Cationic exchange at pH 2.0 selectively removed more biologically produced SON relative to SCOD.

Mass spectroscopic investigation of nucleic acid degradation products

The possibilities of structure elucidation of nucleic acid degradation products and of sequencing of oligonucleotides are discussed.

Nucleases of the oriental hornet ( Vespa orientalis) venom sac extract—I. Acid, neutral and alkaline deoxyribonucleases and their pharmacological effects on cat blood in vitro

Acid, neutral and alkaline deoxyribonucleases and their pharmacological effects on cat blood in vitro. Toxicon 14, 427–433, 1976.—We discovered the presence of several deoxyribonucleases (DNases) in venom sac extracts (VSE) of the oriental hornet ( Vespa orientalis). The DNases were partially purified by gel filtration of G-75 Sephadex column and were tested for their substrate specificities on native, single-stranded and u.v.-irradiated DNA. The DNases consist of both endonucleases and exonucleases. Acid DNases were not inhibited by EDTA, which was inhibitory however, to both neutral and alkaline DNases. Cat blood was treated with venom sac extract (VSE) of the oriental hornet and showed an increase in nucleic acid degradation products. These degradation products may be due to the presence of nucleases in VSE.

Protein Degradation in Escherichia coli: II. STRAIN DIFFERENCES IN THE DEGRADATION OF PROTEIN AND NUCLEIC ACID RESULTING FROM STARVATION

Abstract Based on leucine exchange measurements of cells labeled with [14C]leucine, we had previously reported (J. Biol. Chem., 245, 2889 (1970)) that only a limited class of of Escherichia coli B is subject to a first order process of rapid degradation. The rate of degradation (half-life of 60 min or faster) and the total amount of undergoing degradation (2 to 7%) was the same during growth and during various kinds of starvation. We extend this finding and report here that the release of this degrading protein is unaltered not only during growth and starvation but also during growth in an enriched medium (step-up), in a poor medium (step-down), and during the diauxic phase of induced β-galactosidase synthesis. The extent of this observed degradation (less than 10%) is lower than reported by other workers (20 to 35%). This discrepancy is not a consequence of experimental manipulation or cellular damage but reflects differences in bacterial strains. Although strains ML and K-12 under conditions of growth release radioactivity similar in amount and half-life to the rapid degradation process observed in strain B, during several conditions of starvation, degradation of an additional class of cellular can be measured by leucine exchange in these two strains but not in B. The starvation-induced degradation occurs at a rate of 2.5 to 6% per hour, and the amount of the cellular that degrades as a result of starvation amounts to 20 to 40% of the total bacterial protein. Simultaneous with this starvation-induced degradation is the excretion of nucleic acid degradation products into the medium amounting to 40 to 60% of the ordinarily stable nucleic acid of growing cells. Starvation-induced nucleic acid degradation occurs in all strains equally. With the findings of these strain differences, much of the conflict in the literature can be explained satisfactorily. We conclude that a continuous process of degradation occurs in bacterial cells at all times, and an additional process of degradation concomitant with nucleic acid degradation is initiated following starvation of nutrients. Some of the properties of the degradation processes under starvation conditions are as follows. While almost all of the degradation products released in the presence of carrier leucine are acid soluble, a large portion of nucleic acid degradation products released from the cells are precipitable by cold trichloroacetic acid. Both and nucleic acid degradation occur under starvation of either glucose, nitrogen, or phosphate. Inhibition of synthesis by chloramphenicol at 100 µg per ml inhibits the starvation-induced degradation, but does not affect the degradation of the rapidly degrading protein.

Loss of macrophages from peritoneal exudates of sensitized rats after injection of streptococcal antigens.

Experiments are reported describing the macrophage disappearance phenomenon elicited by streptococcal cell wall antigens in rats sensitized with viable <i>β</i>-hemolytic streptococci emulsified in Freund’s complete adjuvant. Macrophage loss from peritoneal exudates of rats sensitized to group A streptococci was demonstrable after i.p. injection of <i>Streptomyces albus</i> enzyme- or formamide-extracted and not further purified group A antigens. No macrophage disappearance occurred when the same antigens were injected into non-sensitized animals or animals sensitized with streptococci of groups B, C, E or G. In contrast to the chemically extracted partially purified carbohydrate preparations, chromatographically purified C-carbohydrates did not induce macrophage loss. Data are presented which indicate that carbohydrate-mucopeptide complexes and possibly nucleic acid degradation products are responsible for the macrophage disappearance phenomena observed.

Actinomycin D before and during primary and secondary anti-Forssman immunoglobulin hemolysin responses in rabbits.

The effects of actinomycin D were studied on successive stages of the primary and secondary (anamnestic) anti-Forssman immunoglobulin hemolysin responses induced in rabbits by the antigen, sheep red blood cells. (Fully developed anamnestic reactivity was tested 1.5-2 months after the start of an untreated initial response.) The stages of both responses include induction by antigen, the latent period, the acute rise of hemolysin to peak titer, and the decline afterwards. This antibiotic modified both responses significantly in ways strikingly similar to those produced by large doses of x-rays. It was injected in a sublethal amount (0.08 mg/kg) at selected times before or after a test intravenous injection of 10(8.3) sheep erythrocytes/kg. Given within several days before the antigen, it delayed and depressed peak titer, whereas given during induction and the latent period, it delayed and enhanced peak titer. Given during the acute rise, its effect decreased, and given during the decline after peak titer, it only produced erratic slight rises in titer. It is suggested that delayed and depressed peak titers are associated with both known activities of sublethal doses of actinomycin D, i.e., cytotoxicity and inhibition of RNA synthesis, whereas enhanced peak titers are brought about by the presence of nucleic acid degradation products released by the cytotoxicity of actinomycin D at the critical time of active RNA synthesis in the latent period. Effects of actinomycin D differed quantitatively in the two responses. During the initial response, delayed depression was not apparent as soon and did not last as long, whereas delayed enhancement was more pronounced. In terms of the cells operating in the two responses, the data indicate that immunologically competent initial cells, as compared to memory cells, are more easily stimulated, are not injured as quickly, recover more rapidly, and overcompensate for the injury for a longer time. In addition, in untreated controls, recruitment of immunologically competent initial cells appears to be largely inhibited during induction of memory cells. Otherwise, decline of the secondary response would be less abrupt.

Nucleic acid degradation products of Streptomyces aureofaciens.

In industrial fermentations of Streptomyces aureofaciens, accumulation of hypoxanthine, cytosine and guanosine in the fermentation medium depending on the level of chlortetracycline production was observed. A similar dependance was found also in fermentation media of low- and high-producing strains of Streptomyces aureofaciens under laboratory conditions. Chlortetracycline supplemented to the medium for the cultivation of a low- and high-productive strains of S. aureofaciens in an amount of 200 and 500 μg/ml resulted in an increase of the hypoxanthine and cytosine levels. At the same time AMP and CMP in some cases were also found in the medium. Cadaverine added to the fermentation medium in an amount of 0.1 % decreased the level of cytosine by 60% and increased the guanosine content by 50% as compared with the control. The hypoxanthine level remained unchanged. In the acid-soluble fraction of the mycelium of S. aureofaciens nucleotides, nucleosides, and bases with predominance of AMP and its degradation products were found. In the fermentation medium and in the acid-soluble fraction of the mycelium, some unknown ultraviolet-absorbing substances were observed.

PHOSPHATE, PURINES AND PYRIMIDINES AS EXCRETORY PRODUCTS OF TETRAHYMENA.

SYNOPSIS. Tetrahymena pyriformis strain W, incubated for 3 hours in a salts‐PO4 medium, showed a net loss of phosphate from the cell. This loss of phosphate was accompanied by release of material having an absorption maximum at 250–260 nvi. The u.v.‐absorbing material contained hypoxanthine as well as a pyrimidine component.The release of hypoxanthine was consistent with an absence of detectable xanthine oxidase activity in Tetrahymena homogenates. Studies with homogenates indicated that hypoxanthine formation was paralleled by the formation of perchloric acid‐soluble material absorbing at 250 mμ, suggesting the degradation of nucleic acid.While glucose diminished the extent of phosphate and hypoxanthine loss from intact cells, the addition of iodoacetic acid (IAA) resulted in increased losses, as well as release of pentose into the medium. Since IAA did not influence the amount of acid‐soluble material formed by homogenates, it is suggested that the effects of glucose and IAA on the extent of excretion represent alterations in the ability of the cells to utilize nucleic acid degradation products in RXA synthesis.

The effect of calcium sulfate as the binder in deae-cellulose thin-layer chromatography for separating nucleic acid degradation products

Thin-layer techniques involving layers of diethylaminoethyl-cellulose, with and without CaSO4, are presented for the complete separation and quantitative elution of the major degradation products of ribonucleic acid and deoxyribonucleic acid. Emphasis is given to the effects of the pH of the material to be separated and to the percent CaSO4 present in the cellulose as “binder”.

Separation of Nucleic Acid Degradation Products on Partition Columns

Separation of Nucleic Acid Degradation Products on Partition Columns

Nucleic Acid-Splitting Enzymes in Human Epidermis and their Possible Role in Keratinization

Disappearance of nuclei from cells undergoing cornification is one of the most characteristic features of the keratinization process. As an indication of nuclear breakdown, water-soluble degradation products of nucleic acids have been demonstrated in keratinized structures.

Quantitative chromatographic methods. 8. Chromatographic systems of high resolving power for nucleotides.

Two paper chromatographic systems of improved resolving power for nucleotides are described. Both systems are based on buffered miscible solvents, used with chromatographic papers previously impregnated with the same buffers. The potential usefulness of these systems in studies on naturally occurring soluble nucleotides and on degradation products of nucleic acids is illustrated.

Effects of irradiation on metabolism of nucleic acids and the acid soluble derivatives in rats

In the rat, X-irradiation leads to a decrease of the net RNA and DNA content of the spleen, without, however, increasing the low molecular weight nucleic acid derivatives in the acid-soluble fractions of the same tissue. It is suggested that an alteration of the cell membranes could account for the apparent elimination of these nucleic acid degradation products which should normally appear in the acid-soluble fraction.

Some new solvent systems for the paper chromatography of nucleic acid degradation products

Some new solvent systems for the paper chromatography of nucleic acid degradation products