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At least 1,063 records · Page 59Linked to original sources
PubMed1988

Factors affecting possible carcinogenicity of inhaled nitropyrene aerosols.

Nitroaromatics in general, and 1-nitropyrene in particular, are potent bacterial mutagens and animal carcinogens. Their importance as possible human carcinogens is difficult to assess because they are usually found in the environment as the products of combustion processes, and so they usually exist with many other compounds associated with airborne particles. The experiments reported here were carried out to determine if the inhalation of particle-associated 1-nitropyrene, or the concomitant exposure to an irritant gas, would alter the tissue distribution of 1-nitropyrene or its metabolites, compared to their distribution after inhalation of pure 1-nitropyrene. These experiments were intended to yield insights into the mechanisms involved in the potential carcinogenicity of particle-associated nitroaromatics as inhaled in the environment from automotive emissions and other sources. Groups of Fischer 344 rats inhaled pure 14C-1-nitropyrene aerosols, with and without coexposure to 5 parts per million sulfur dioxide, or 14C-1-nitropyrene adsorbed onto gallium oxide particles, with and without coexposure to sulfur dioxide, for four weeks. Lung retention of 14C-1-nitropyrene was not prolonged by its association with gallium oxide particles or by coexposure to sulfur dioxide. There was a marked inflammatory and fibrogenic response to the gallium oxide particles. Another set of experiments was carried out in which rats were exposed to 14C-1-nitropyrene either as pure aerosol or adsorbed onto carbon black particles. The amount of 14C in the lung that was bound to carbon black particles steadily decreased with time after exposure, compared to total lung 14C, indicating removal of 14C from the particles. Thirty minutes after exposure, the amount of 14C covalently bound to lung macromolecules, expressed as a percentage of calculated deposited radioactivity, was twofold greater for 1-nitropyrene adsorbed onto carbon black than for 1-nitropyrene alone. The amount of covalently bound 14C increased with time after exposure to 14C-1-nitropyrene adsorbed onto carbon black, reaching a level of approximately 1 percent of deposited radioactivity, 10-fold greater than that seen with pure 14C-1-nitropyrene seven to 30 days after exposure. The level of covalently bound 14C declined steadily after exposure to pure 14C-1-nitropyrene. Carbon black particles associated with adsorbed 1-nitropyrene offer the potential of studying DNA adduct formation in the lung, because DNA modification might be greater after inhalation of 1-nitropyrene adsorbed onto carbon black than after inhalation of pure 1-nitropyrene or 1-nitropyrene associated with metal oxides, such as gallium oxide.(ABSTRACT TRUNCATED AT 400 WORDS)

Air Pollutants↗
PubMedDate not supplied

[EcoRV restrictase: physical and catalytic properties of homogenous enzyme].

With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity. The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons. According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII. Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied. It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place. It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification. DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro. Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack. The fragments resulted may be cloned in canonical pBR322 EcoRV site.

Base Sequence↗
PubMed1983

[Role of the oncogenes and the protein kinases in carcinogenesis].

The genome of higher animals contains genes called cOnc which are thought to be responsible for cancer when activated. Many oncogenic retroviruses also contain vOnc genes responsible for their transforming properties. cOnc sequences are homologous and result, in fact, from genetic recombinations between retroviruses lacking transforming genes and the animal genome. Onc genes encode transforming proteins which are biological intermediates of their carcinogenic property and may consist of protein kinases active on membrane and cytoskeleton constituents or nuclear proteins probably active on DNA. Modifications of cOnc genes (or oncogenes) due, in particular, to chromosomal rearrangements and occasional mutations have been demonstrated in an increasing number of human cancers.

Animals↗
PubMedDate not supplied

[Affinity modification of DNA-dependent RNA-polymerase of phage T7 with 5'-p-fluorosulfonylbenzoyladenosine].

The affinity modification of the DNA-dependent RNA-polymerase of bacteriophage T7 was carried out by using the specific irreversible inhibitor, 5'-p-fluorosulfonylbenzoyladenosine. The inhibitor was found to bind to the enzyme's active site; the kinetic constants of the modification were calculated. The stoichiometry of the covalent E.I-complex formed was determined by using the 14C-labeled inhibitor.

Adenosine↗
PubMed2006

N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage.

The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N(6)-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3'-formylphosphate residues arising from 5'-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N(6)-amino groups of lysine side chains. A liquid chromatography (LC)-tandem mass spectrometry (MS) method was developed to quantify the resulting N(6)-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04-0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose-response relationship for the formation of N(6)-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N(6)-formylation of the linker histone H1. The N(6)-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N(6)-acetylation recognized as an important determinant of gene expression in mammalian cells. The N(6)-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress.

Acetylation↗
PubMed2006

DNA-methyltransferase SsoII as a bifunctional protein: features of the interaction with the promoter region of SsoII restriction-modification genes.

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.

Catalytic Domain↗
PubMedDate not supplied

[Affinity modification of DNA-dependent RNA-polymerase from phage T7 with 5'-p-fluorosulfonylbenzoyl adenosine: the effect of modification on the interaction with substrates].

T7 RNA polymerase, covalently modified with 5'-p-fluorosulfonylbenzoyl adenosine, looses the ability of binding the promoter (pGEM-2 plasmid) and poly(dC) template as well as the initiating nucleoside triphosphate (GTP). However the enzyme retains the unspecific binding with DNA fragments of considerable length.

Adenosine↗
PubMed2007

DNA methylation and histone modifications in patients with cancer: potential prognostic and therapeutic targets.

Epigenetics, a combination of DNA modifications, chromatin organization, and variations in its associated proteins, configure a new entity that regulates gene expression throughout methylation, acetylation, and chromatin remodeling. In addition to silencing as a result of mutations, loss of heterozygosity, or classical genetic events epigenetic modification symbolizes essential early events during carcinogenesis and tumor development. The reversion of these epigenetic processes restoring normal expression of tumor-suppressor genes has consequently become a new therapeutic target in cancer treatment. Aberrant patterns of epigenetic modifications will be, in a near future, crucial parameters in cancer diagnosis and prognosis.

Acetylation↗
PubMed1991

The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.

Amino Acid Sequence↗
PubMed2000

Human T lymphocyte genetic modification with naked DNA.

Endowing T lymphocytes with novel functional attributes by genetic modification is under development for a broad range of clinical cellular immunotherapy applications. To circumvent many of the limitations associated with viral vector systems, a plasmid-based electroporation system that reliably generates G418-resistant primary human T lymphocyte clones was developed. TCR alpha/beta+ CD4+CD8-, and CD4-CD8+ T lymphocyte clones can be routinely isolated from OKT3-stimulated peripheral blood mononuclear cells electroporated with linear plasmid DNA in a limiting dilution drug selection format. Fluorescence in situ hybridization (FISH) studies performed on T cell metaphase spreads using a probe specific for plasmid sequence demonstrated a single FISH signal doublet that varied in chromosomal location from clone to clone. Southern blot analysis using a Neo-specific probe verified chromosomal integration of plasmid vector at a single site. Band intensity quantitation of blots developed with a zeta-specific probe capable of annealing to both endogenous TCR-zeta and the introduced chimeric zeta sequence demonstrated that integrated plasmid was present at a single copy number. Expression levels of the CD20-specific chimeric immunoreceptor construct from a CMV immediate/early promoter present in the plasmid vector varied widely from clone to clone but remained stable during ex vivo expansion to cell numbers in excess of 10(10). This T lymphocyte genetic modification strategy is currently being piloted in a FDA-sanctioned adoptive therapy trial for recurrent lymphoma.

CD4-Positive T-Lymphocytes↗
PubMedDate not supplied

[Selective modification of T7 DNA at the region of early genes by early RNA carrying multiple alkylating groups].

A method of selective modification of certain regions of the genome which may become useful for inactivation of certain genes or for directed mutagenesis is proposed. For this purpose RNA products of certain genes carrying alkylating groupings randomly distributed along the polymer were used. The RNA modified to an extent of 4--5 alkylating residues per 100 nucleotides retains the ability to specific formation of DNA--RNA hybrid complexes. The alkylating molecule is N,N,N'-tri-(beta-chlorethyl), N'-(p-formylphenyl)propylene diamine-1,3. The aliphatic alkylating functions serve for attachment to RNA. The aromatic alkylating function inactivated by the formyl grouping at the para-position of the benzene ring is used for modification of DNA after hybrid formation by reduction of formyl grouping with sodium borohydride. The covalently binding of modified RNA is exhibited to occur in only the case of T7 DNA H-chain, the one complementary to the RNA derivative. L-chain does not hybridize, nor does it undergo alkylation by the RNA product thus indicating high selectivity of alkylation within the hybrid complex.

Coliphages↗
PubMed2002

N4,5-dimethylcytosine, a novel hypermodified base in DNA.

Successive enzymatic modification of DNA by two methyltransferases that recognize identical or overlapping sequences and normally target either 5- or N4-position of the same cytosine residue, can lead to the formation of a doubly methylated base--N4,5-dimethylcytosine. Implications of such enzymatic "permethylation" of cytosine on the structure and interactions of DNA are examined.

Cytosine↗
PubMed2005

Complicated tails: histone modifications and the DNA damage response.

In recent years, several ATP-dependent chromatin-remodeling complexes and covalent histone modifications have been implicated in the response to double-stranded DNA breaks (DSBs). When a DSB occurs, cells must identify the DSB, activate the DNA damage checkpoint, and repair the break. Chromatin modification appears to be important but not essential for each of these processes, yet its precise mechanistic roles are only beginning to come into focus. Here, we discuss the role of chromatin in signaling by the DNA damage checkpoint pathway.

Animals↗
PubMed1998

Modification of kinetoplast DNA minicircle composition in pentamidine-resistant Leishmania.

Pentamidine, an antiprotozoal drug, was shown to have various cellular and molecular targets depending on the organism. In Leishmania, ultrastructural modifications of kinetoplast and mitochondria have been observed but no data is available on cellular and molecular events involved in development of pentamidine-resistance. The absence of modification of minicircle DNA in pentamidine treated L. donovani and L. amazonensis promastigotes suggested that topoisomerase II activity is not a target. This result was confirmed by quantitation of the enzyme by immunodetection. Southern blot experiments indicated that the kDNA network was altered in resistant clones. Molecular cloning and sequence analysis of kDNA minicircles showed transkinetoplastidy hitherto reported only for arsenite- and tunicamycin-resistant Leishmania. Comparison of wild-type and resistant sequences showed only 32-51% homology. The AT-rich regions, known as binding sites, of the drug occurred less frequently in the resistant clones and their locations were different. These minicircle sequence modifications leading to decreased binding sites for the drug might contribute to pentamidine-resistance in Leishmania.

Animals↗
PubMed1987

[Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. IX. Cleavage of substrates with point modifications in the recognition site and flanking sequences].

Ability of the EcoRII restriction endonuclease to cleave 14-base-pair DNA duplexes with nucleotide substitutions in the recognition site CCA/TGG and in the adjacent base pair has been studied. Modifications leading to a local change in the substrate conformation (rU residue in and outside the recognition site, A.A- or A.C-pairs in the flanking sequence) reduce the rate of hydrolysis, the effect being maximal when the modified base pair is outside the recognition site. No digestion occurs when the internal dC-residue of the recognition site is 5-methylated in one or both strands. Replacement of dT residue in the EcoRII recognition site by dfl5U residue results in a dramatic inhibition of hydrolysis. Km and kcat for the cleavage of 14-base-pair DNA duplex have been determined. The cleavage rate of the dT-containing strand of the recognition site in 1.5 fold higher comparing with the dA-containing strand. The cleavage of both strands of the substrate by EcoRII endonuclease is confirmed to proceed in one enzyme-substrate complex.

Base Sequence↗
PubMed2026

Mapping Protein Occupancy on DNA with an Unnatural Cytosine Modification.

The epigenome provides a dynamic layer of gene regulatory control above the static genetic sequence. DNA base modifications are key epigenetic regulators, predominantly found within CpG contexts in mammalian genomes. Working in tandem with these DNA modifications, chromatin-associated proteins and transcription factors further control gene expression. Given the interplay of these factors, concurrent mapping of DNA base modifications with protein-DNA occupancy can greatly aid in interpreting the epigenome. Existing multimodal mapping methods include the use of DNA methyltransferases to mark accessible, protein-unbound DNA in non-CpG contexts. However, such approaches can either confound readouts with native DNA modifications or constrain users to third-generation sequencing approaches. To circumvent these limitations, we explored the possibility of introducing an unnatural DNA base modification, 5-carboxymethylcytosine, as an alternative label for protein occupancy. Here, we report our efforts to rationally engineer non-CpG-specific DNA methyltransferases to take on neomorphic DNA carboxymethyltransferase (CxMTase) activities. We find that DNA carboxymethylation of cytosines in GpC contexts shows broad compatibility with the most widely used epigenetic detection methods and can be used to reliably report on protein occupancy states. Using this approach, we reveal the single-molecule binding patterns of LexA, a master repressor in the bacterial DNA damage (SOS) response, at its self-regulated and endogenously methylated promoter. We thus show that unnatural DNA modifications can uncover novel biological insights and potentiate new approaches to multimodal epigenetic profiling.

DNA↗
PubMed1995

Early biochemical markers of effects: enzyme induction, oncogene activation and markers of oxidative damage.

Experimental carcinogenicity studies focus on identification of single carcinogens. Humans, however, appear exposed to a variety of low doses of carcinogens. Furthermore, few chemical entities are carcinogenic or toxic per se, but require metabolic activation to form ultimate carcinogens or toxins. In contrast to experimental animals, humans show considerable difference in genetic properties. In that situation it is particularly important to estimate individual capability for metabolic activation. To an increasing extent, activation includes formation of toxic oxygen metabolites. Particular targets for activated species are DNA and lipids; in particular low-density lipoproteins (LDL). Modifications of DNA are important for initiating the multistep process of carcinogenesis, in particular if oncogenes are activated or if tumor supressor genes are inactivated. Such DNA modification can be identical regardless of the reactive specimens being a xenobiotic or an oxygen species. Modification of LDL can start the process of atherosclerosis by transforming macrophages into foam cells, deposited as fatty streaks in the arterial wall. Biomarkers for activation capacity of xenobiotics include the use of prototype substrates and molecular techniques to determine genetic polymorphisms. Oxidative DNA modification can be measured from urinary excretion of oxidatively modified deoxynucleosides, particularly guanosine. Future efforts have to include individual measurements in order to improve the 'resolution' of molecular epidemiological approaches.

8-Hydroxy-2'-Deoxyguanosine↗