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Model of adduct formation by alkylating GMO in the presence of RecA. The alkylating agent (nitrogen mustard) and linker arm are depicted in black. Strands of the target duplex DNA are shown in different shades of gray. The darker strand is complementary to the GMO and the lighter strand is homologous to the GMO. RecA protein coats the GMO and catalyzes strand invasion. An alkylation reaction tethers the GMO to the target.
Source publication
Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides
(ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mut...
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The role of glutathione-S-transferase (GST) in alkylator drug resistance has been studied in MatB rat mammary carcinoma cells. A series of GST transfectant cell lines was established by using an expression vector containing the complementary DNA for the rat GST Yc gene under regulation of the SV40 early region promoter and the antibiotic resistance...
Citations
... The nitrogen mustard family are used as chemotherapeutic agents against multiple myeloma, lymphoma, leukemia, ovarian carcinoma and various other tumors (Balcome et al., 2004). Similarly to psoralens, nitrogen mustards conjugated to TFO can be used for a targeted gene modification in mammals (Singer et al., 1999). Experimentally, the most commonly used nitrogen mustard is bis(2-chloroethyl)methylamine (HN2, mechlorethamine), a bifunctional alkylating agent. ...
DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermediates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.
... Again the majority of damage consists of monoadducts to the DNA, however, the 1–5% ICLs are responsible for the high cytotoxicity (22). Oligonucleotides conjugated to nitrogen mustards can be used to introduce ICLs at specific sites in the genome (23). ...
Impaired DNA damage repair, especially deficient transcription-coupled nucleotide excision repair, leads to segmental progeroid
syndromes in human patients as well as in rodent models. Furthermore, DNA double-strand break signalling has been pinpointed
as a key inducer of cellular senescence. Several recent findings suggest that another DNA repair pathway, interstrand cross-link
(ICL) repair, might also contribute to cell and organism aging. Therefore, we summarize and discuss here that (i) systemic
administration of anti-cancer chemotherapeutics, in many cases DNA cross-linking drugs, induces premature progeroid frailty
in long-term survivors; (ii) that ICL-inducing 8-methoxy-psoralen/UVA phototherapy leads to signs of premature skin aging
as prominent long-term side effect and (iii) that mutated factors involved in ICL repair like ERCC1/XPF, the Fanconi anaemia
proteins, WRN and SNEV lead to reduced replicative life span in vitro and segmental progeroid syndromes in vivo. However, since ICL-inducing drugs cause damage different from ICL and since all currently known ICL repair factors work
in more than one pathway, further work will be needed to dissect the actual contribution of ICL damage to aging.
... The nitrogen mustard family are used as chemotherapeutic agents against multiple myeloma, lymphoma, leukemia, ovarian carcinoma and various other tumors (Balcome et al., 2004). Similarly to psoralens, nitrogen mustards conjugated to TFO can be used for a targeted gene modification in mammals (Singer et al., 1999). Experimentally, the most commonly used nitrogen mustard is bis(2-chloroethyl)methylamine (HN2, mechlorethamine), a bifunctional alkylating agent. ...
DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermedi-ates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.
This article provides an overview of how functional genomics is likely to impact on the pathology laboratory and highlights how informatics and tissue banking will greatly facilitate the molecular age of medicine. Important aspects of functional genomics in the post-genome era, including the roles of laser capture microdissection, DNA- and complementary DNA-based microarrays, proteomic methods, collaborative human tissue banking, tissue microarrays, and pathobioinformatics in the modern pathology laboratory are discussed. The role of mass spectroscopy in the analysis of RNA, DNA, and protein and its impact on the clinical laboratory, particularly in cost-effectiveness and time savings, are evaluated. This article explores how laboratory information systems (LISs) and the devices that feed them information may need to be modified to adapt to greater volumes of data for the new testing modalities that require understanding sophisticated fluorescence detection methods and image processing. Emerging genomic testing methods and their impact on pathology laboratory testing, especially in the area of molecular classification of neoplasms, are examined. The role of the tissue bank in the modern pathology laboratory as an archive of control normal tissues, as well as subsamples of the spectrum of progressive neoplastic states, is discussed in light of its critical importance to the molecular classification of cancer. Establishing a database that combines structured reports in pathology LISs and construction of tissue banking information systems will provide a rich resource for pathology departments. The article discusses a hypothetical resource, such as the Shared Tumor Expression Profiler, that would provide access to well-characterized tissue-based research resources for clinicians and researchers. Last, the article emphasizes how LISs can prepare for these changes, and how training pathologists in pathology informatics and bioinformatics (pathobioinformatics) is critical to ensure pathology’s overall leadership role in the post-genome era.
The Yessentukskoye deposit of Caucasian mineral waters contains balneologically valuable drinking mineral water, which is extracted from the Upper Cretaceous 1 km subsurface aquifer and is almost unexplored by microbiologists. We have sampled this water via continuously operating production wells, characterized the phylogenetic diversity of its microbial community, and obtained enrichments of thermophilic iron reducers from the source aquifer. From the enrichments, a novel anaerobic thermophilic bacterium, reducing Fe(III) in the mineral ferrihydrite with acetate as the electron donor, was isolated into a pure culture. The novel isolate, designated as strain Es71-Z0220 T belonging to Deferribacterales order, is thermophilic, neutrophilic, halotolerant, motile vibrio. It utilizes synthesized ferrihydrite, fumarate, nitrate or elemental sulfur as the electron acceptors with organic acids as the electron donors. The strain is incapable of soluble Fe(III) complexes reduction and fermentative growth. The draft genome assembly of strain Es71-Z0220 T resulted in 65 contigs with a total size of ca. 2.3 Mb. On the basis of whole-genome phylogenetic reconstruction and physiological characterization, the novel isolate was considered to represent a novel family, genus and species for which the name Deferrivibrio essentukiensis gen. nov., sp. nov. is proposed. Genome analysis revealed key determinants of anaerobic respiration and carbon substrate utilization pathways in the organism with peculiarities related to putative Fe(III)-reducing electron transfer chain. Considering the revealed metabolic features of Deferrivibrio essentukiensis , the involvement of the organism in its subsurface environment in biogeochemical by carbon cycling by coupling the organic matter oxidation with Fe(III) minerals reduction is discussed.
The formation of a fluorescent photoadduct between 5‐fluoro‐4‐thiouridine (FS U), in the sequence context 5′‐AFS UA‐3′ and incorporated into a synthetic oligonucleotide either at its 3′‐ or 5′‐end, and one of the thymines of the TAT motif in a complementary target DNA strand led to photo‐crosslinking of the two strands for several oligonucleotide constructs. Enzymatic digestion, MS, UV, and fluorescence spectral analyses of the interstrand crosslinked oligonucleotides revealed the identity of the thymidine that participates in the photo‐crosslinking reaction as well as the diastereomeric structures of the crosslinks. The proposed pathways of interstrand photo‐crosslinking are supported by experiments with isotopically labeled oligonucleotide constructs and visualized by means of molecular dynamics simulations. Always cross(link) at the light: Selective photoexcitation of 5‐fluoro‐4‐thio‐2′‐O‐methyluridine (FS U) between two adenosines in oligonucleotide duplexes results in highly efficient crosslinking of the two strands by formation of a fluorescent adduct (P) with thymidine.
The highest incidences of cancer are found in the skin, but endogenous pigmentation is associated with markedly reduced risk. Agents that enhance skin pigmentation have the potential to reduce both photodamage and skin cancer incidence. The purpose of this review is to evaluate agents that have the potential to increase skin pigmentation. These include topically applied substances that simulate natural pigmentation: dihydroxyacetone and melanins; and, substances that stimulate the natural pigmentation process: psoralens with UVA (PUVA), dimethylsulfoxide (DMSO), l-tyrosine, l-Dopa, lysosomotropic agents, diacylglycerols, thymidine dinucleotides, DNA fragments, melanocyte stimulating hormone (MSH) analogs, 3-isobutyl-1-methylxanthine (IBMX), nitric oxide donors, and bicyclic monoterpene (BMT) diols. These agents are compared with regard to efficacy when administered to melanoma cells, normal human epidermal melanocytes, animal skin, and human skin. In addition, mechanisms of action are reviewed since these often reveal issues releated to both efficacy and safety. Both dihydroxyacetone and topically applied melanins are presently available to the consumer, and both of these have been shown to provide some photoprotection. Of the pigmentation stimulators, only PUVA and MSH analogs have been tested extensively on humans, but there are concerns about the safety and side effects of both. At least some of the remaining pigmentation stimulators under development have the potential to safely induce a photoprotective tan.
This article provides an overview of how functional genomics is likely to impact on the pathology laboratory and highlights how informatics and tissue banking will greatly facilitate the molecular age of medicine. Important aspects of functional genomics in the post-genome era, including the roles of laser capture microdissection, DNA- and complementary DNA-based microarrays, proteomic methods, collaborative human tissue banking, tissue microarrays, and pathobioinformatics in the modern pathology laboratory are discussed. The role of mass spectroscopy in the analysis of RNA, DNA, and protein and its impact on the clinical laboratory, particularly in cost-effectiveness and time savings, are evaluated. This article explores how laboratory information systems (LISs) and the devices that feed them information may need to be modified to adapt to greater volumes of data for the new testing modalities that require understanding sophisticated fluorescence detection methods and image processing. Emerging genomic testing methods and their impact on pathology laboratory testing, especially in the area of molecular classification of neoplasms, are examined. The role of the tissue bank in the modern pathology laboratory as an archive of control normal tissues, as well as subsamples of the spectrum of progressive neoplastic states, is discussed in light of its critical importance to the molecular classification of cancer. Establishing a database that combines structured reports in pathology LISs and construction of tissue banking information systems will provide a rich resource for pathology departments. The article discusses a hypothetical resource, such as the Shared Tumor Expression Profiler, that would provide access to well-characterized tissue-based research resources for clinicians and researchers. Last, the article emphasizes how LISs can prepare for these changes, and how training pathologists in pathology informatics and bioinformatics (pathobioinformatics) is critical to ensure pathology's overall leadership role in the post-genome era.
As the 21st century moves forward, it is becoming more and more apparent that the genetic makeup of any individual strongly influences the way they metabolise nutrients. It is very important, therefore, to understand the techniques and technologies used to assess the contribution genes make to the physiology of an individual. Clearly, it is not possible to provide a comprehensive overview, but in the present review an attempt will be made to show, using examples from the authors' research, how these methods have contributed to this understanding. Studies are being undertaken into Fe transport across the placenta, from the mother to the fetus, and the consequences of maternal anaemia on pregnancy outcome. Levels of gene transcript and protein have been measured using Northern and Western blotting respectively. During the course of this work a new protein has been identified using the available human genome database. Following this 'in silico' or 'cyber biology', techniques such as real-time RT-PCR and RNA interference have been used to examine expression of this gene and its protein. The methods used, briefly how they work and some of their limitations will be explained. The objective of the present review is primarily to give a better perception of how molecular biology can be used in research and to help gain a clearer understanding of some of the techniques used.
We have found that a short stretch (30mer or larger) of triple-stranded DNA structure formed at the terminus (or very near)
of linear DNA molecules is unusually stable, withstanding heat treatment at as high as 95°C. The stable triple-stranded structure
is formed only when deoxyoligonucleotides are complementary to the strand terminating with 5′-phosphate and not to the strand
terminating with 3′-OH. Presence of a single mismatched base in the complementary deoxyoligonucleotides drastically reduces
the stability. We show that these unique properties of the terminal triple-stranded structure can be applied to the detection
of single nucleotide polymorphisms in genomes without DNA dissociation and/or hybridization.
