Improved Tensor Current Limit from

A precision measurement of the ß^{+} decay of ^{8}B was performed using the Beta-decay Paul Trap to determine the ß-ν angular correlation coefficient a_{ßν}. The experimental results were combined with new ab initio symmetry-adapted no-core shell-model calculations to yield the second-most precise measurement from Gamow-Teller decays, a_{ßν}=-0.3345±0.0019_{stat}±0.0021_{syst}. This value agrees with the standard model value of -1/3 and improves uncertainties in ^{8}B by nearly a factor of 2. By combining results from ^{8}B and ^{8}Li, a tight limit on tensor current coupling to right-handed neutrinos was obtained. A recent global evaluation of all other precision ß decay studies suggested a nonzero value for right-handed neutrino coupling in contradiction with the standard model at just above 3σ. The present results are of comparable sensitivity and do not support this finding.

Direct Determination of Fission-Barrier Heights Using Light-Ion Transfer in Inverse Kinematics.

We demonstrate a new technique for obtaining fission data for nuclei away from ß stability. These types of data are pertinent to the astrophysical r process, crucial to a complete understanding of the origin of the heavy elements, and for developing a predictive model of fission. These data are also important considerations for terrestrial applications related to power generation and safeguarding. Experimentally, such data are scarce due to the difficulties in producing the actinide targets of interest. The solenoidal-spectrometer technique, commonly used to study nucleon-transfer reactions in inverse kinematics, has been applied to the case of transfer-induced fission as a means to deduce the fission-barrier height, among other variables. The fission-barrier height of ^{239}U has been determined via the ^{238}U(d,pf) reaction in inverse kinematics, the results of which are consistent with existing neutron-induced fission data indicating the validity of the technique.

Quenching of Single-Particle Strength in A=15 Nuclei.

Absolute cross sections for the addition of s- and d-wave neutrons to ^{14}C and ^{14}N have been determined simultaneously via the (d,p) reaction at 10 MeV/u. The difference between the neutron and proton separation energies, ΔS, is around -20 MeV for the ^{14}C+n system and +8 MeV for ^{14}N+n. The population of the 1s_{1/2} and 0d_{5/2} orbitals for both systems is reduced by a factor of approximately 0.5 compared with the independent single-particle model, or about 0.6 when compared with the shell model. This finding strongly contrasts with results deduced from intermediate-energy knockout reactions between similar nuclei on targets of ^{9}Be and ^{12}C. The simultaneous technique used removes many systematic uncertainties.

Search for Nova Presolar Grains: γ-Ray Spectroscopy of

The discovery of presolar grains in primitive meteorites has initiated a new era of research in the study of stellar nucleosynthesis. However, the accurate classification of presolar grains as being of specific stellar origins is particularly challenging. Recently, it has been suggested that sulfur isotopic abundances may hold the key to definitively identifying presolar grains with being of nova origins and, in this regard, the astrophysical ^{33}Cl(p,γ)^{34}Ar reaction is expected to play a decisive role. As such, we have performed a detailed γ-ray spectroscopy study of ^{34}Ar. Excitation energies have been measured with high precision and spin-parity assignments for resonant states, located above the proton threshold in ^{34}Ar, have been made for the first time. Uncertainties in the ^{33}Cl(p,γ) reaction have been dramatically reduced and the results indicate that a newly identified ℓ=0 resonance at E_{r}=396.9(13) keV dominates the entire rate for T=0.25-0.40 GK. Furthermore, nova hydrodynamic simulations based on the present work indicate an ejected ^{32}S/^{33}S abundance ratio distinctive from type-II supernovae and potentially compatible with recent measurements of a presolar grain.

Key

Detection of nuclear-decay γ rays provides a sensitive thermometer of nova nucleosynthesis. The most intense γ-ray flux is thought to be annihilation radiation from the ß^{+} decay of ^{18}F, which is destroyed prior to decay by the ^{18}F(p,α)^{15}O reaction. Estimates of ^{18}F production had been uncertain, however, because key near-threshold levels in the compound nucleus, ^{19}Ne, had yet to be identified. We report the first measurement of the ^{19}F(^{3}He,tγ)^{19}Ne reaction, in which the placement of two long-sought 3/2^{+} levels is suggested via triton-γ-γ coincidences. The precise determination of their resonance energies reduces the upper limit of the rate by a factor of 1.5-17 at nova temperatures and reduces the average uncertainty on the nova detection probability by a factor of 2.1.

Constraint of the astrophysical

The Galactic 1.809-MeV γ-ray signature from the ß decay of ^{26g}Al is a dominant target of γ-ray astronomy, of which a significant component is understood to originate from massive stars. The ^{26g}Al(p,γ)^{27}Si reaction is a major destruction pathway for ^{26g}Al at stellar temperatures, but the reaction rate is poorly constrained due to uncertainties in the strengths of low-lying resonances in ^{27}Si. The ^{26g}Al(d,p)^{27}Al reaction has been employed in inverse kinematics to determine the spectroscopic factors, and hence resonance strengths, of proton resonances in ^{27}Si via mirror symmetry. The strength of the 127-keV resonance is found to be a factor of 4 higher than the previously adopted upper limit, and the upper limit for the 68-keV resonance has been reduced by an order of magnitude, considerably constraining the ^{26g}Al destruction rate at stellar temperatures.

Halo nucleus 11Be: a spectroscopic study via neutron transfer.

The best examples of halo nuclei, exotic systems with a diffuse nuclear cloud surrounding a tightly bound core, are found in the light, neutron-rich region, where the halo neutrons experience only weak binding and a weak, or no, potential barrier. Modern direct-reaction measurement techniques provide powerful probes of the structure of exotic nuclei. Despite more than four decades of these studies on the benchmark one-neutron halo nucleus 11Be, the spectroscopic factors for the two bound states remain poorly constrained. In the present work, the 10Be(d,​p) reaction has been used in inverse kinematics at four beam energies to study the structure of 11Be. The spectroscopic factors extracted using the adiabatic model were found to be consistent across the four measurements and were largely insensitive to the optical potential used. The extracted spectroscopic factor for a neutron in an nℓj=2s(1/2) state coupled to the ground state of 10Be is 0.71(5). For the first excited state at 0.32 MeV, a spectroscopic factor of 0.62(4) is found for the halo neutron in a 1p(1/2) state.

Mitochondrial DNA damage is involved in apoptosis caused by pro-inflammatory cytokines in human OA chondrocytes.

OBJECTIVE: Pro-inflammatory cytokines play a pivotal role in cartilage destruction during the progression of osteoarthritis (OA). Additionally, these cytokines are capable to generate reactive oxygen and nitrogen species within chondrocytes. Mitochondrion is a prime target of oxidative damage and an important player in aging and degenerative processes. The purpose of the present study was to investigate whether these cytokines will alter the mitochondrial DNA (mtDNA) integrity and mitochondrial function in both normal and osteoarthritic human chondrocytes. DESIGN: Primary normal and osteoarthritic human chondrocyte cultures were exposed to various concentrations of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) for different time. Following exposure, chondrocytes were evaluated for mitochondrial DNA damage, ATP production, changes in mitochondrial transcription, and apoptosis. Adenoviral vectors were used to deliver DNA repair enzyme hOGG1 to mitochondria. RESULTS: Pro-inflammatory cytokines IL-1beta and TNF-alpha disturb mitochondrial function in human chondrocytes by inducing mitochondrial DNA damage, decreasing energy production and mitochondrial transcription, which correlated with the induction of apoptosis. Increased NO production was the key factor responsible for accumulation of mtDNA damage after cytokine exposure. Mitochondrial superoxide production was also enhanced following pro-inflammatory cytokine exposure. OA chondrocyte mitochondria were more susceptible to damage induced by pro-inflammatory cytokines then mitochondria from normal chondrocytes. Protection of human chondrocytes from mtDNA damage by the mitochondria-targeted DNA repair enzyme hOGG1 rescued mtDNA integrity, preserved ATP levels, reestablished mitochondrial transcription, and significantly diminished apoptosis following IL-1beta and TNF-alpha exposure. CONCLUSION: Mitochondrion is an important target in pro-inflammatory cytokine toxicity, maintaining of mitochondrial DNA integrity is necessary to prevent chondrocytes from apoptosis induced by IL-1beta and TNF-alpha.

Diminished mitochondrial DNA integrity and repair capacity in OA chondrocytes.

OBJECTIVES: Osteoarthritis (OA) is characterized by the failure of chondrocytes to respond to injury and perform the cartilage remodeling process. Human articular chondrocytes actively produce reactive oxygen and nitrogen species (ROS and RNS) capable of causing cellular dysfunction and death. A growing body of evidence indicates that mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage play a causal role in disorders linked to excessive generation of oxygen free radicals. The aim of this study was to determine whether mtDNA damage was present in OA chondrocytes, and whether mtDNA repair capacity is compromised in OA chondrocytes following oxidative stress, leading to chondrocyte death. METHODS: Human articular cartilage was isolated from knee joints of cadavers available through the Anatomical Gifts Program at the University of South Alabama (normal donors) or OA patients undergoing total knee replacement surgeries (OA patients). Total DNA was isolated from either chondrocytes released following collagenase digestion, or from first passage chondrocytes grown in culture and exposed to ROS or RNS. mtDNA integrity and repair capacity were analyzed by quantitative Southern blot analysis, using a mtDNA-specific radioactive probe. Cell viability was determined by the trypan blue exclusion method. RESULTS: mtDNA damage was found in chondrocytes from OA patients compared to normal donors. It was accompanied with reduced mtDNA repair capacity, cell viability, and increased apoptosis in OA chondrocytes following exposure to ROS and RNS. CONCLUSIONS: These results indicate that mtDNA damage and poor mtDNA repair capacity for removing damage caused by oxidative stress may contribute to the pathogenesis of OA.

Mitochondrial dysfunction and delayed hepatotoxicity: another lesson from troglitazone.

AIMS/HYPOTHESIS: Troglitazone was approved for treatment of type 2 diabetes mellitus, but by 2000 it had been removed from all world markets due to severe drug-induced liver injury. Even today, we still do not know how many patients sustained a long-term liver injury. No system is in place to acquire that knowledge. Regarding toxicity mechanisms, controversy persists as to which ones are class effects of thiazolidinediones (TZDs) and which are unique to troglitazone. This study aims to provide long-term outcome data and new insights on mechanisms of troglitazone-induced liver injury. METHODS: This case series reports the liver injuries sustained by eleven type 2 diabetic patients treated with troglitazone between 1997 and 2000. Exhaustive review of medical records was performed for all patients. Long-term outcomes were available for all the non-fatal cases. A comprehensive literature review was also performed. RESULTS: Long-term liver injury progressing to cirrhosis was identified in seven patients. All eleven cases had liver injury patterns consistent with troglitazone toxicity. Analysis of these cases and of the experimental troglitazone toxicity data points to mitochondrial toxicity as a central factor. The general clinical patterns of mitochondrial hepatotoxic events are reviewed, as are the implications for other members of the TZD family. CONCLUSIONS/INTERPRETATION: This analysis enables the liver injury induced by troglitazone to be better understood. In future cases of delayed drug-induced liver injury that progresses after discontinuation, the possibility of mitochondrial toxicity should be considered. When appropriate, this can then be evaluated experimentally. Such proactive investigation may anticipate clinical risk before a large-scale therapeutic misadventure occurs. Drug-induced liver injury due to mitochondrial hepatotoxins may be less unpredictable than has previously been surmised.

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes.

Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitochondrial DNA (mtDNA). At present, only palliative therapies are available for these disorders, and interest in the development of efficient treatment protocols is high. Here, we demonstrate that in cells heteroplasmic for the T8993G mutation, which is a cause for the NARP and MILS syndromes, infection with an adenovirus, which encodes the mitochondrially targeted R.XmaI restriction endonuclease, leads to selective destruction of mutant mtDNA. This destruction proceeds in a time- and dose-dependent manner and results in cells with significantly increased rates of oxygen consumption and ATP production. The delivery of R.XmaI to mitochondria is accompanied by improvement in the ability to utilize galactose as the sole carbon source, which is a surrogate indicator of the proficiency of oxidative phosphorylation. Concurrently, the rate of lactic acid production by these cells, which is a marker of mitochondrial dysfunction, decreases. We further demonstrate that levels of phosphorylated P53 and gammaH2ax proteins, markers of nuclear DNA damage, do not change in response to infection with recombinant adenovirus indicating the absence of nuclear DNA damage and the relative safety of the technique. Finally, some advantages and limitations of the proposed approach are discussed.

Mitochondrial DNA repair: a critical player in the response of cells of the CNS to genotoxic insults.

Cells of the CNS are constantly exposed to agents which damage DNA. Although much attention has been paid to the effects of this damage on nuclear DNA, the nucleus is not the only organelle containing DNA. Within each cell, there are hundreds to thousands of mitochondria. Within each mitochondrion are multiple copies of the mitochondrial genome. These genomes are extremely vulnerable to insult and mutations in mitochondrial DNA (mtDNA) have been linked to several neurodegenerative diseases, as well as the normal process of aging. The principal mechanism utilized by cells to avoid DNA mutations is DNA repair. Multiple pathways of DNA repair have been elucidated for nuclear DNA. However, it appears that only base excision repair is functioning in mitochondria. This repair pathway is responsible for the removal of most endogenous damage including alkylation damage, depurination reactions and oxidative damage. Within the rat CNS, there are cell-specific differences mtDNA repair. Astrocytes exhibit efficient repair, whereas, other glial cell types and neuronal cells exhibit a reduced ability to remove lesions from mtDNA. Additionally, a correlation was observed between those cells with reduced mtDNA repair and an increase in the induction of apoptosis. To demonstrate a causative relationship, a strategy of targeting DNA repair proteins to mitochondria to enhance mtDNA repair capacity was employed. Enhancement of mtDNA repair in oligodendrocytes provided protection from reactive oxygen species- and cytokine-induced apoptosis. These experiments provide a novel strategy for protecting sensitive CNS cells from genotoxic insults and thus provide new treatment options for neurodegenerative diseases.

Palmitate induced mitochondrial deoxyribonucleic acid damage and apoptosis in l6 rat skeletal muscle cells.

A major characteristic of type 2 diabetes mellitus (T2DM) is insulin resistance in skeletal muscle. A growing body of evidence indicates that oxidative stress that results from increased production of reactive oxygen species and/or reactive nitrogen species leads to insulin resistance, tissue damage, and other complications observed in T2DM. It has been suggested that muscular free fatty acid accumulation might be responsible for the mitochondrial dysfunction and insulin resistance seen in T2DM, although the mechanisms by which increased levels of free fatty acid lead to insulin resistance are not well understood. To help resolve this situation, we report that saturated fatty acid palmitate stimulated the expression of inducible nitric oxide (NO) synthase and the production of reactive oxygen species and NO in L6 myotubes. Additionally, palmitate caused a significant dose-dependent increase in mitochondrial DNA (mtDNA) damage and a subsequent decrease in L6 myotube viability and ATP levels at concentrations as low as 0.5 mM. Furthermore, palmitate induced apoptosis, which was detected by DNA fragmentation, caspase-3 cleavage, and cytochrome c release. N-acetyl cysteine, a precursor compound for glutathione formation, aminoguanidine, an inducible NO synthase inhibitor, and 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III), a peroxynitrite inhibitor, all prevented palmitate-induced mtDNA damage and diminished palmitate-induced cytotoxicity. We conclude that exposure of L6 myotubes to palmitate induced mtDNA damage and triggered mitochondrial dysfunction, which caused apoptosis. Additionally, our findings indicate that palmitate-induced mtDNA damage and cytotoxicity in skeletal muscle cells were caused by overproduction of peroxynitrite.

Protection of human keratinocyte mtDNA by low-level nitric oxide.

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the beta-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.

Base excision repair of mitochondrial DNA damage in mammalian cells.

This review of the work from our laboratory describes initial studies in which base excision repair in mtDNA was first seen. It considers the results of experiments in which the substrates for mtDNA repair were identified. The discussion then focuses on studies during which the sequence context for mtDNA damage and repair were explored. Next, it addresses factors that have been identified that influence mtDNA repair. Finally, it summarizes the results of studies that evaluated cell-specific differences in the repair of mtDNA and explored some of the biological consequences of these differences.

Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes.

Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly repaired in MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA lesions at nucleotide resolution showed that damage patterns were similar between EC phenotypes, but there were disparities between XO and menadione in terms of the specific nucleotides damaged. These findings indicate that mtDNA in lung vascular ECs is damaged by XO- and menadione-derived free radicals and suggest that mtDNA damage and repair capacities differ between EC phenotypes.

Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts.

Transcription-coupled repair (TCR) plays an important role in removing DNA damage from actively transcribed genes. It has been speculated that TCR is the most important mechanism for repairing DNA damage in non-dividing cells such as neurons. Therefore, abnormal TCR may contribute to the development of many age-related and neurodegenerative diseases. However, the molecular mechanism of TCR is not well understood. Oligonucleotide DNA triplex formation provides an ideal system to dissect the molecular mechanism of TCR since triplexes can be formed in a sequence-specific manner to inhibit transcription of target genes. We have recently studied the molecular mechanism of triplex-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extracts. Using plasmid constructs we demonstrate that the level of TFO-mediated DNA repair activity is directly correlated with the level of transcription of the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity was further linked with transcription since the presence of rNTPs in the reaction was essential for AG30-mediated DNA repair activity in HeLa nuclear extracts. The involvement of individual components, including TFIID, TFIIH, RNA polymerase II and xeroderma pigmentosum group A (XPA), in the triplex-mediated TCR process was demonstrated in HeLa nuclear extracts using immunodepletion assays. Importantly, our studies also demonstrated that XPC, a component involved in global genome DNA repair, is involved in the AG30-mediated DNA repair process. The results obtained in this study provide an important new understanding of the molecular mechanisms involved in the TCR process in mammalian cells.

Poly(ADP-ribose) polymerase facilitates the repair of N-methylpurines in mitochondrial DNA.

This study was designed to test the hypothesis that poly(ADP-ribose) polymerase (PARP) plays a role in the repair of damage to mitochondrial DNA (mtDNA). A rat insulinoma cell line was transfected with a PARP antisense vector that was under the control of a dexamethasone promoter. Transfected cells were selected for stable integration of the antisense vector. Several cell lines containing the antisense vector were isolated. For these studies, one of these lines (clone 5) was chosen for further evaluation. When cells were treated with dexamethasone for 72 h, PARP activity was diminished by 60%. Western blot analysis revealed a concomitant reduction in PARP protein. When clone 5 cells were exposed to the simple methylating agent methylnitrosourea (MNU) without previous treatment with dexamethasone, repair of lesions in mtDNA was found to be similar to that seen in wild-type cells or in wild-type cells treated with dexamethasone. However, when clone 5 cells were pretreated with dexamethasone for 72 h, repair of MNU-induced damage was significantly inhibited. To ascertain whether the PARP activity that was inhibited by the antisense treatment was the same as that found in the nucleus, repair studies were performed on fibroblasts derived from PARP knockout mice and their normal wild-type controls. Attenuated repair was also seen in the cells in which the gene for PARP was inactivated. These are the first studies to demonstrate that PARP can facilitate the repair of simple alkylation damage to mtDNA.

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology.

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.