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Impact of copper on the induction and repair of oxidative DNA damage, poly(ADP-ribosyl)ation and PARP-1 activity.

Copper is an essential trace element involved, among other functions, in enzymatic antioxidative defense systems. However, nonprotein bound copper ions have been shown to generate reactive oxygen species. To gain insight into the discrepancy between the protective properties of copper on the one hand and its toxicity on the other hand, we examined the genotoxic effects of CuSO(4) in cultured human cells. Here we report that copper, at cytotoxic concentrations, induces oxidative DNA base modifications and DNA strand breaks. However, at lower noncytotoxic concentrations, copper inhibits the repair of oxidative DNA damage induced by visible light. As a first mechanistic hint, inhibition of H(2)O(2)-induced poly(ADP-ribosyl)ation was identified in cultured cells and further experiments demonstrated a strong inhibition of the activity of isolated poly(ADP-ribose)polymerase-1 (PARP-1) by copper. Bioavailability studies of copper showed a dose-dependent uptake in cells and pointed out the relevance of the applied concentrations. Taken together, the results indicate that copper, under conditions of either disturbed homeostasis or overload due to high exposure, exerts defined genotoxic effects. Hence, a balance needs to be maintained to ensure sufficient uptake and to prevent overload.

Copper↗

The appearance of new structures and functions in proteins during evolution.

The likelihood of a de novo generation of classes of efficient proteins through neoformation of DNA, through modification of expressed DNA, and through modification of nonexpressed DNA is examined. So is the likelihood that newly formed inefficient enzymes be turned into efficient enzymes. The conclusions are that neither gene duplicates nor dormant genes represent promising materials for a de novo generation of protein classes, that (with exceptions) such generation is unlikely to have taken place in recent evolution, that new structural genes must nearly consistently derive from preexisting structural genes, and that new functions can be evolved only on the basis of old proteins. Conditions of protein evolution in prokaryotes suggest that the saltatory formation of protein classes is as unlikely in prokaryotes as in eukaryotes. Data on the history of a few protein classes are reviewed to illustrate the preceding inferences. The analysis leads to the hypothesis that most protein classes originated before the major elements of the translation apparatus of modern cells were fully evolved. If simple sequence DNA is turned into structural genes by evolution, this process (again with exceptions) is considered to have taken place only at that very remote period. A polyphyletic origin of proteins is thought to date back to the same era. It is proposed that the development of genic multiplicity and of marked structural and functional diversity of proteins may have come about in the earliest cells primarily through the independent generation of structurally different polymerases in different protocells, followed by cell conjugation and the subsequent use by enriched cells of supernumerary types of polymerase for evolving further functions. Functional growth, as it took place at early times, is briefly discussed as well as functional change. The foundations for new functional developments in old proteins are analyzed. In considering the evolutionary recovery of lost functions, aspects of cell differentiation and gene regulation are linked with the evolutionary picture. The distinction between eurygenic and stemogenic control of gene activity is used. Next to gene deletion, cell and tissue deletion is held to be an event of general evolutionary significance, through cell and tissue origination that presumably accompanies the restoration of a lost molecular function.

Amino Acids↗

Molecular mechanisms of quinone cytotoxicity.

Quinones are probably found in all respiring animal and plant cells. They are widely used as anticancer, antibacterial or antimalarial drugs and as fungicides. Toxicity can arise as a result of their use as well as by the metabolism of other drugs and various environmental toxins or dietary constituents. In rapidly dividing cells such as tumor cells, cytotoxicity has been attributed to DNA modification. However the molecular basis for the initiation of quinone cytotoxicity in resting or non-dividing cells has been attributed to the alkylation of essential protein thiol or amine groups and/or the oxidation of essential protein thiols by activated oxygen species and/or GSSG. Oxidative stress arises when the quinone is reduced by reductases to a semiquinone radical which reduces oxygen to superoxide radicals and reforms the quinone. This futile redox cycling and oxygen activation forms cytotoxic levels of hydrogen peroxide and GSSG is retained by the cell and causes cytotoxic mixed protein disulfide formation. Most quinones form GSH conjugates which also undergo futile redox cycling and oxygen activation. Prior depletion of cell GSH markedly increases the cell's susceptibility to alkylating quinones but can protect the cell against certain redox cycling quinones. Cytotoxicity induced by hydroquinones in isolated hepatocytes can be attributed to quinones formed by autoxidation. The higher redox potential benzoquinones and naphthoquinones are the most cytotoxic presumably because of their higher electrophilicty and thiol reactivity and/or because the quinones or GSH conjugates are more readily reduced to semiquinones which activate oxygen.

Animals↗

On the meaning of fragile sites in cancer risk and development.

In the last few years, there has been increasing concern about the possible involvement of fragile sites in cancer risk and development. Patients with malignancies and family histories of cancer who presented with constitutional fragile sites are reported here. These findings are discussed with regard to the familial risk for cancer and the tissue specificity of the malignancy in relation to the different fragile sites. The hypothesis is advanced that these may be sites of viral DNA modification, probably representing areas where genes that are important for the metabolism of the virus are located. On the other hand, these genes may well be cellular (proto)oncogenes. We believe that fragile sites may increase the risk for cancer, not by being break-prone points at oncogene locations, but through more complex mechanisms that are not easy to predict.

Chromosome Banding↗

The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function.

The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene. Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters. The com gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDal, has been visualized on gels. Efficient expression of Mom from pL requires gene com. A fusion between MS-2 polymerase and com has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage mu↗

Post-transcriptional regulation of the bacteriophage Mu mom gene by the com gene product.

The mom gene of bacteriophage Mu encodes a DNA modification function, the expression of which is detrimental to the host cell. This may be reflected by the tight regulation of the mom gene at the level of transcription initiation by the Mu C gene product and the host Dam function. In addition, mom expression requires the positive regulatory function Com. The com and mom genes comprise the mom operon with the com coding region partially overlapping that of mom. The degree of overlap is defined by experiments reported here. We have tested Com for activity as an antiterminator of mom transcription. We show that in the absence of Com, premature termination affects at most 33% of the transcription across the mom operon. Although no premature termination is observed in the presence of Com, these results are inconsistent with a role for Com as an antiterminator. Northern blot analysis of Com+ and Com- Mu phage mRNA confirms this conclusion. Two models for the post-transcriptional regulation of mom gene expression by Com are presented.

Adenine↗

Determination of hemoglobin adducts of arylamines in humans.

Aromatic amines and nitroarenes are important occupational and environmental pollutants. It is therefore essential to have fast and reliable methods to monitor human exposure. In rat experiments it has been shown that several arylamines form hydrolyzable hemoglobin adducts. Hemoglobin adducts are dosimeters for the internal dose and possibly for DNA modification at the site of tumor formation. In the present paper we introduce a new efficient method to quantify hemoglobin adducts. Precipitated hemoglobin was hydrolyzed in base in the presence of the recovery standards d5-aniline, d4-2-methylaniline, d4-3-methylaniline, d4-4-methylaniline, d3-2,4-dimethylaniline, 13C6-4-chloroaniline, d9-4-aminobiphenyl, and 4'-fluoro-4-aminobiphenyl. The hydrolysate was extracted with hexane, derivatized with pentafluoropropionic anhydride (PFPA) and analyzed by GC-MS with negative chemical ionization. In one run over twenty amines can be determined. The method has been applied to human studies.

Amines↗

Mdm2 is critically and continuously required to suppress lethal p53 activity in vivo.

There is currently much interest in the idea of restoring p53 activity in tumor cells by inhibiting Hdm2/Mdm2. However, it has remained unclear whether this would also activate p53 in normal cells. Using a switchable endogenous p53 mouse model, which allows rapid and reversible toggling of p53 status between wild-type and null states, we show that p53 is spontaneously active in all tested tissues of mdm2-deficient mice, triggering fatal pathologies that include ablation of classically radiosensitive tissues. In apoptosis-resistant tissues, spontaneous unbuffered p53 activity triggers profound inhibition of cell proliferation. Such acute spontaneous p53 activity occurs in the absence of any detectable p53 posttranslational modification, DNA damage, or p19ARF signaling and triggers rapid p53 degradation.

ADP-Ribosylation Factor 1↗

Therapeutic and preventive interventions for postulated vasoactive neuropeptide autoimmune fatigue-related disorders.

Major advances have been made in understanding the relatively novel group of vasoactive (vasodilatory) neuropeptides (VNs) in humans. VNs comprise a novel but expanding group of substances having immunoregulation, inflammation modulation, neurotransmitter, neurotrophic, hormonal and metabolic functions. These substances may control gene expression for mRNA for themselves and their receptors. They have complex relationships with gaseous and other neurotransmitters and xenobiotic substances. Theoretical arguments have implicated these substances in autoimmune phenomena resulting in fatigue-related conditions such as chronic fatigue syndrome (CFS), sudden infant death syndrome (SIDS), fibromyalgia (FM) and Gulf War syndrome (GWS) but remain unproven. As well as possibly spontaneous onset, the precipitating causes of VN autoimmune dysfunction are likely to be a combination of genetic predisposition, infection and xenobiotic substances. Therapeutic and preventive possibilities for postulated VN autoimmune conditions will be influenced by the complex patholophysiology underpinning them. Some speculative possibilities are VN substitution/replacement, preservation of biological effect, epigenetic DNA modifications, plasma exchange, anti-cholinesterases, e.g., pyridostigmine, corticosteroids and other drug treatments, thymectomy, intravenous immunoglobulin and anti-idiotype antibodies, and CpG/DNA vaccines. Prevention and treatment of possible VN autoimmune fatigue-related disorders may prove to be important areas for future research and development.

Autoimmune Diseases↗

Mutagens in surface waters: a review.

A review of the literature on the mutagenicity/genotoxicity of surface waters is presented in this article. Subheadings of this article include a description of sample concentration methods, mutagenic/genotoxic bioassay data, and suspected or identified mutagens in surface waters published in the literature since 1990. Much of the published surface water mutagenicity/genotoxicity studies employed the Salmonella/mutagenicity test with strains TA98 and/or TA100 with and/or without metabolic activation. Among all data analyzed, the percentage of positive samples toward TA98 was approximately 15%, both in the absence and the presence of S9 mix. Those positive toward TA100 were 7%, both with and without S9 mix. The percentage classified as highly mutagenic (2500-5000 revertants per liter) or extremely mutagenic (more than 5000 revertants per liter) was approximately 3-5% both towards TA98 and TA100, regardless of the absence or the presence of S9 mix. This analysis demonstrates that some rivers in the world, especially in Europe, Asia and South America, are contaminated with potent direct-acting and indirect-acting frameshift-type and base substitution-type mutagens. These rivers are reported to be contaminated by either partially treated or untreated discharges from chemical industries, petrochemical industries, oil refineries, oil spills, rolling steel mills, untreated domestic sludges and pesticides runoff. Aquatic organisms such as teleosts and bivalves have also been used as sentinels to monitor contamination of surface water with genotoxic chemicals. DNA modifications were analyzed for this purpose. Many studies indicate that the 32P-postlabeling assay, the single cell gel electrophoresis (comet) assay and the micronucleus test are sensitive enough to monitor genotoxic responses of indigenous aquatic organisms to environmental pollution. In order to efficiently assess the presence of mutagens in the water, in addition to the chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs. This is because according to this review they proved to be sensitive and reliable tools in the detection of mutagenic activity in aquatic environment. Many attempts to identify the chemicals responsible for the mutagenicity/genotoxicity of surface waters have been reported. Among these reports, researchers identified heavy metals, PAHs, heterocyclic amines, pesticides and so on. By combining the blue cotton hanging method as an adsorbent and the O-acetyltransferase-overproducing strain as a sensitive strain for aminoarenes, Japanese researchers identified two new type of potent frameshift-type mutagens, formed unintentionally, in several surface waters. One group has a 2-phenylbenzotriazole (PBTA) structure, and seven analogues, PBTA-type mutagens, were identified in surface waters collected at sites below textile dyeing factories and municipal wastewater treatment plants treating domestic wastes and effluents. The other one has a polychlorinated biphenyl (PCB) skelton with nitro and amino substitution group and it was revealed to be 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl derived from chemical plants treating polymers and dye intermediates. However, the identification of major putative mutagenic/genotoxic compounds in most surface waters with high mutagenic/genotoxic activity in the world have not been performed. Further efforts on chemical isolation and identification by bioassay-directed chemical analysis should be performed.

Asia↗

Fructose 1,6-bisphosphate aldolase activity in leaves of a rice mutant selected for enhanced lysine.

Unknown proteins isolated from mutant tissues of rice (Oryza sativa L.) recovered from inhibitor selections were subsequently peptide microsequenced. Database searches putatively identified one peptide as fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Tissues of mutant rice, PI564784, and wild type (cv Calrose 76) tissues were evaluated for aldolase activity. Total enzyme activities were slightly lower in the mutant than the control but the differences were not significant. Although the mutant phenotype is for enhanced lysine and protein, we ascribe the small aldolase differences to physiological adjustments, rather than to DNA modifications of the aldolase gene(s). Homologies of rice peptides with aldolases from a range of species, as well as rice cell culture expressed sequence tags (ESTs) are presented. Some amino acids sequences are highly conserved. The mutant phenotype expressing stress proteins is not likely to be defined by a change in rice aldolases.

Amino Acid Sequence↗

Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.

Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.

APOBEC-1 Deaminase↗

Immunogenicity of DNA damaged by reactive oxygen species--implications for anti-DNA antibodies in lupus.

Reactive oxygen species (ROS) are implicated in the inflammatory, autoimmune, connective tissue disease, systemic lupus erythematosus (SLE), particularly in respect of processes leading to the formation of pathological anti-DNA antibodies. Exposure to ROS increases the antigenicity of DNA for SLE antibodies, but data on the immunogenicity of ROS-DNA are not conclusive. In this study, we have examined the immunogenicity in rabbits, of DNA modified by three hydroxyl radical generating systems. Additionally, we investigated the antigenicity of UVA, UVB, and UVC irradiated DNA for lupus anti-DNA antibodies. Modification of DNA by both ROS and far UV dramatically increased its immunogenicity; the Fe2+ and H2O2 system resulted in antibodies that recognized both native and modified DNA. In our ELISA system, none of the UV antigens showed any antigenicity above native DNA for SLE sera. The data suggested that different profiles of antigenicity and immunogenicity arise dependent on the method of ROS production, but also that ROS-DNA may be a factor in antigen-driven immune complex formation in SLE.

Adult↗

Maintenance of chromatin states: an open-and-shut case.

The traditional view of chromatin envisions two states: one is 'active' and accessible to nucleases, whereas the other is 'silent' and relatively inaccessible. Recent evidence that combinations of diverse histone tail modifications represent a spectrum of chromatin states challenges this simple view. Here, we examine inter-relationships between chromatin remodeling, histone modification, DNA methylation, RNA interference, and nucleosome assembly activities. We find that the two-state view can accommodate these new findings, and that nucleosome assembly pathways may ultimately maintain euchromatic and heterochromatic states.

Animals↗

Herpesvirus genetics has come of age.

The genetic analysis of the large and complex herpesviruses has been a constant challenge to herpesvirologists. Elegant methods have been developed to produce mutants in infected cells that rely on the cellular recombination machinery. Bacterial artificial chromosomes (BACs), single copy F-factor-based plasmid vectors of intermediate insert capacity, have now enabled the cloning of complete herpesvirus genomes. Infectious virus genomes can be shuttled between Escherichia coli and eukaryotic cells. Herpesvirus BAC DNA engineering in E. coli by homologous recombination requires neither restriction sites nor cloning steps and allows the introduction of a wide variety of DNA modifications. Such E. coli-based technology has provided a safe, fast and effective approach to the systematic mining of the information stored in herpesvirus genomes as a result of their intimate co-evolution with their specific hosts for millions of years. Use of this technique could lead to new developments in clinical virology and basic virology research, and increase the usage of viral genomes as investigative tools and vectors.

Animals↗

Listening to the silent genes: transgene silencing, gene regulation and pathogen control.

By capitalizing on the initially puzzling observations of unpredictable transgene silencing and variable expression, plant scientists have pioneered research into a novel type of epigenetic regulation, termed homology-dependent gene silencing. This silencing process has implications for natural mechanisms of gene expression in plants and other eukaryotes, and has branched out into studies of reversible DNA modifications; RNA metabolism, transport and processing; and host responses to plant viruses, viroids and transposable elements. The analysis of transgene silencing systems has enriched our understanding of other epigenetic phenomena, including paramutation, as well as heterosis and genome evolution. This research is also highly relevant to the biotechnology industry, which is interested in avoiding unwanted transgene silencing in genetically engineered lines and in exploiting various types of silencing to inactivate specific genes. Homology-dependent gene silencing can also be used in high-throughput approaches for functional genomics.

Journal Article↗

Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution.

The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com.

Bacteriophage mu↗

Mechanistic Information on the Redox Cycling of Nickel(II/III) Complexes in the Presence of Sulfur Oxides and Oxygen. Correlation with DNA Damage Experiments.

The reactions of the water-soluble complexes [NiCR](2+) (where CR = 2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15-pentaene) and [NiKGH-CONH(2)](+) (where KGH-CONH(2) = lysylglycylhistidinecarboxamide) with sulfite/O(2) and peroxymonosulfate have been investigated using spectrophotometric and rapid-scan techniques. In most cases, the spectral changes suggest the formation of an intermediate Ni(III) species, followed by decomposition reactions which involve a back-reaction to Ni(II). Only in the case of the [NiCR](2+)-S(IV)-O(2) system is the formed Ni(III) species stable in solution. When sulfite and oxygen are used to oxidize Ni(II) to Ni(III), the reaction is oxygen dependent and an induction period could be observed, whereas the use of the strong oxidizing agent peroxymonosulfate resulted in no induction period and no oxygen dependence. In addition, the oxidation of Ni(II) to Ni(III) was faster if peroxymonosulfate was used instead of sulfite/O(2). The [NiKGH-CONH(2)](+) complex reacts much faster with sulfite/O(2) and peroxymonosulfate than the [NiCR](2+) does. Rate constants for the oxidation process and possible reaction mechanisms, based on available literature data, that can account for the observed kinetic observations in a qualitative way are presented, and the results are correlated with previously obtained data on DNA modification using these systems.

Journal Article↗