Thrombin generation: a positive or negative response to trauma in dogs?
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Animals↗
Biomedical subjects
R D Wood
Publications and source records attributed to R D Wood.
Increasing the magnetic helicity content of a plasma by pulsing a magnetized source.
By operating a magnetized coaxial gun in a pulsed mode it is possible to produce large voltage pulses of duration approximately 500 mus while reaching a few kV, giving a discrete input of helicity into a spheromak. In the sustained spheromak physics experiment (SSPX), it is observed that pulsing serves to nearly double the stored magnetic energy and double the temperature. We discuss these results by comparison with 3D MHD simulations of the same phenomenon.
Journal Article↗
New mode of operating a magnetized coaxial plasma gun for injecting magnetic helicity into a spheromak.
By operating a magnetized coaxial plasma gun continuously with just sufficient current to enable plasma ejection, large gun-voltage spikes (approximately 1 kV) are produced, giving the highest sustained voltage approximately 500 V and highest sustained helicity injection rate observed in the Sustained Spheromak Physics Experiment. The spheromak magnetic field increases monotonically with time, exhibiting the lowest fluctuation levels observed during formation of any spheromak (B/B>/=2%). The results suggest an important mechanism for field generation by helicity injection, namely, the merging of helicity-carrying filaments.
Journal Article↗
Suppression of MHD fluctuations leading to improved confinement in a gun-driven spheromak.
Magnetic fluctuations have been reduced to approximately 1% during discharges on the Sustained Spheromak Physics Experiment by shaping the spatial distribution of the bias magnetic flux in the device. In the resulting quiescent regime, the safety factor profile is nearly flat in the plasma and the dominant ideal and resistive MHD modes are greatly reduced. During this period, the temperature profile is peaked at the magnetic axis and maps onto magnetic flux contours. Energy confinement time is improved over previous reports in a driven spheromak.
Journal Article↗
Numerical simulation of the superplastic forming of dental and medical prostheses.
A relatively recent development in prostheses manufacture is the use of superplastic forming technology formerly associated with industrial applications such as aerospace components. This paper considers the finite element incremental flow formulation for the simulation of the forming of thin and moderately thick titanium alloy sheet dental and medical prostheses and, in particular, the determination of the pressure-time cycle. Issues concerning material characteristics are discussed, and the process is illustrated by a number of examples. The application of finite element simulation in this context is ongoing, nevertheless interim conclusions are drawn and future directions for research are identified.
Biocompatible Materials↗
Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.
Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.
DNA Damage↗
Human DNA repair genes.
Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process.
DNA↗
Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair.
ERCC1-XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5' to 3' away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1-XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1-XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.
Amino Acid Sequence↗
A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum.
Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.
Amino Acid Sequence↗
Emerging links between hypermutation of antibody genes and DNA polymerases.
Substantial antibody variability is created when nucleotide substitutions are introduced into immunoglobulin variable genes by a controlled process of hypermutation. Evidence points to a mechanism involving DNA repair events at sites of targeted breaks. In vertebrate cells, there are many recently identified DNA polymerases that inaccurately copy templates. Some of these are candidates for enzymes that introduce base changes during hypermutation. Recent research has focused on possible roles for DNA polymerases zeta (POLZ), eta (POLH), iota (POLI), and mu (POLM) in the process.
Animals↗
Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome.
In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.
DNA Repair↗
Disruption of the developmentally regulated Rev3l gene causes embryonic lethality.
The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.
Animals↗
Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease.
Interstrand DNA cross-link damage is a severe challenge to genomic integrity. Nucleotide excision repair plays some role in the repair of DNA cross-links caused by psoralens and other agents. However, in mammalian cells there is evidence that the ERCC1-XPF nuclease has a specialized additional function during interstrand DNA cross-link repair, beyond its role in nucleotide excision repair. We placed a psoralen monoadduct or interstrand cross-link in a duplex, 4-6 bases from a junction with unpaired DNA. ERCC1-XPF endonucleolytically cleaved within the duplex on either side of the adduct, on the strand having an unpaired 3' tail. Cross-links that were cleaved only on the 5' side were purified and reincubated with ERCC1-XPF. A second cleavage was then observed on the 3' side. Relevant partially unwound structures near a cross-link may be expected to arise frequently, for example at stalled DNA replication forks. The results show that the single enzyme ERCC1-XPF can release one arm of a cross-link and suggest a novel mechanism for interstrand cross-link repair.
Base Sequence↗
Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites.
Nucleotide excision repair (NER) of DNA damage requires an efficient means of discrimination between damaged and non-damaged DNA. Cells from humans with xeroderma pigmentosum group C do not perform NER in the bulk of the genome and are corrected by XPC protein, which forms a complex with hHR23B protein. This complex preferentially binds to some types of damaged DNA, but the extent of discrimination in comparison to other NER proteins has not been clear. Recombinant XPC, hHR23B, and XPC-hHR23B complex were purified. In a reconstituted repair system, hHR23B stimulated XPC activity tenfold. Electrophoretic mobility-shift competition measurements revealed a 400-fold preference for binding of XPC-hHR23B to UV damaged over non-damaged DNA. This damage preference is much greater than displayed by the XPA protein. The discrimination power is similar to that determined here in parallel for the XP-E factor UV-DDB, despite the considerably greater molar affinity of UV-DDB for DNA. Binding of XPC-hHR23B to UV damaged DNA was very fast. Damaged DNA-XPC-hHR23B complexes were stable, with half of the complexes remaining four hours after challenge with excess UV-damaged DNA at 30 degrees C. XPC-hHR23B had a higher level of affinity for (6-4) photoproducts than cyclobutane pyrimidine dimers, and some affinity for DNA treated with cisplatin and alkylating agents. XPC-hHR23B could bind to single-stranded M13 DNA, but only poorly to single-stranded homopolymers. The strong preference of XPC complex for structures in damaged duplex DNA indicates its importance as a primary damage recognition factor in non-transcribed DNA during human NER.
Animals↗
Removal of oxygen free-radical-induced 5',8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells.
Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5' position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5'R or 5'S form of 5',8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals.
Base Sequence↗
UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome.
Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.
Base Pairing↗
TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair.
TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5' nor 3' incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients.
Adenosine Triphosphatases↗
Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK.
During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase delta or epsilon, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.
Cyclin-Dependent Kinases↗