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A precision measurement of the β^{+} decay of ^{8}B was performed using the Beta-decay Paul Trap to determine the β-ν angular correlation coefficient a_{βν}. 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_{βν}=-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 β 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.

We demonstrate a new technique for obtaining fission data for nuclei away from β 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.

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.

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.

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 β^{+} 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.

Quasifree one-proton knockout reactions have been employed in inverse kinematics for a systematic study of the structure of stable and exotic oxygen isotopes at the R^{3}B/LAND setup with incident beam energies in the range of 300-450  MeV/u. The oxygen isotopic chain offers a large variation of separation energies that allows for a quantitative understanding of single-particle strength with changing isospin asymmetry. Quasifree knockout reactions provide a complementary approach to intermediate-energy one-nucleon removal reactions. Inclusive cross sections for quasifree knockout reactions of the type ^{A}O(p,2p)^{A-1}N have been determined and compared to calculations based on the eikonal reaction theory. The reduction factors for the single-particle strength with respect to the independent-particle model were obtained and compared to state-of-the-art ab initio predictions. The results do not show any significant dependence on proton-neutron asymmetry.

The Galactic 1.809-MeV γ-ray signature from the β 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.

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.

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., (Copyright 2009 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine DNA glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed that this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared with cells transfected with vector only, whereas nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of the tetrazolium compound to formazan, trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.

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.

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., (Copyright 2000 Academic Press.)

Glial cell types in the central nervous system are continuously exposed to reactive oxygen species (ROS) due to their high oxygen metabolism and demonstrate differential susceptibility to certain pathological conditions believed to involve oxidative stress. The purpose of the current studies was to test the hypothesis that mtDNA damage could contribute to the differential susceptibility of glial cell types to apoptosis induced by oxidative stress. Primary cultures of rat astrocytes, oligodendrocytes, and microglia were utilized, and menadione was used to produce the oxidative stress. Apoptosis was detected and quantitated in menadione-treated oligodendrocytes and microglia (but not astrocytes) using either positive annexin-V staining or positive staining for 3'-OH groups in DNA. The apoptotic pathway that was activated involved the release of cytochrome c from the intermitochondrial space and activation of caspase 9. Caspase 8 was not activated after exposure to menadione in any of the cells. Using equimolar concentrations of menadione, more initial damage was observed in mtDNA from oligodendrocytes and microglia. Additionally, using concentrations of menadione that resulted in comparable initial mtDNA damage, more efficient repair was observed in astrocytes compared to either oligodendrocytes or microglia. The differential susceptibility of glial cell types to oxidative damage and apoptosis did not appear related to cellular antioxidant capacity, because under the current culture conditions astrocytes had lower total glutathione content and superoxide dismutase activity than oligodendrocytes and microglia. These results show that the differential susceptibility of glial cell types to menadione-induced oxidative stress and apoptosis appears to correlate with increased oxidative mtDNA damage and support the hypothesis that mtDNA damage could participate in the initiation of apoptosis through the enhanced release of cytochrome c and the activation of caspase 9.

Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutations is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study was designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glycosylase or 3-methyladenine DNA glycosylase failed to reveal additional damage, indicating that most of the lesions produced were caused by the deamination of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the methylating chemical, methylnitrosourea. Most of the lesions produced by NO were repaired rapidly. However, there appeared to be a subset of lesions which were repaired either slowly or not at all by the mitochondria.

Most contemporary progress in Alzheimer's disease (AD) stems from the study of a 42 43 amino acid peptide. called the amyloid beta protein (Abeta), as the main neuropathologic marker of the disorder. It has been demonstrated that Abeta has neurotoxic properties and that such effects are mediated by free-radicals. Exposure of neuronal cells to Abeta results in a spectrum of oxidative lesions that are profoundly harmful to neuronal homeostasis. We had previously shown that Abeta25-35 induces oxidative damage to mitochondrial DNA (mtDNA) and that this modality of injury is prevented by melatonin. Because Abeta25 35 does not occur in AD and because the mode of toxicity by Abeta25-35 may be different from that of Abeta1-42 (the physiologically relevant form of Abeta), we extended our initial observations to determine whether oxidative damage to mtDNA could also be induced by Abeta1-42 and whether this type of injury is prevented by melatonin. Exposure of human neuroblastoma cells to Abeta1-42 resulted in marked oxidative damage to mtDNA as determined by a quantitative polymerase chain reaction method. Addition of melatonin to cell cultures along with Abeta completely prevented the damage. This study supports previous findings with Abeta25-35, including a causative role for Abeta in the mitochondrial oxidative lesions present in AD brains. Most important, the data confirms the neuroprotective role of melatonin in Abeta-mediated oxidative injury. Because melatonin also inhibits amyloid aggregation, lacks toxicity, and efficiently crosses the blood-brain barrier, this hormone appears superior to other available antioxidants as a candidate for pharmacologic intervention in AD.

Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluorescein-conjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time- and energy-dependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin alpha, importin beta, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import.

Recent evidence indicates that oxidative DNA damage may be a major cause of aging. One of the more sensitive targets is the mitochondrial genome which is 10 times more susceptible to mutation than is the nuclear genome. A number of age-related neuromuscular degenerative diseases also have been associated with mutations in mitochondrial DNA (mtDNA), and progressive accumulation of oxidative damage in mtDNA from neuronal tissues over time has been shown. In support of the notion that oxidative stress leads to aging is the finding in Down's syndrome (DS), which is characterized by premature aging, that there is enhanced oxidative stress resulting from the aberrant expression of CuZn superoxide dismutase (CuZn SOD). On the basis of these observations, we hypothesized that there may be defective repair of oxidative damage in mtDNA which would ultimately lead to defective electron transport and concomitant enhanced production of reactive oxygen species (ROS). This effect would heighten the oxidative burden in the cell and accelerate the development of phenotypes associated with aging. To evaluate repair of oxidative damage in mtDNA, fibroblasts from several DS patients were treated with the reactive oxygen generator menadione. Oxidative damage was assessed at 0, 2, and 6 h after exposure using a Southern-blot technique and a mtDNA specific probe. The results of these studies show that DS cells are impaired in their ability to repair oxidative damage to mtDNA compared to age-matched control cells. Therefore, this data supports the possibility that increased production of ROS from mitochondria plays a crucial role in the development of aging phenotypes.

Oligodendrocytes are preferentially sensitive to the toxic, carcinogenic, and teratogenic effects of methylnitrosourea (MNU). The mechanisms responsible for this enhanced sensitivity have not been fully elucidated. One of the most vulnerable cellular targets for this chemical is mitochondrial DNA (mtDNA). To determine if differences in mtDNA damage and repair capacity exist among the different CNS glial cell types, the effects of MNU exposure on oligodendroglia, astroglia, and microglia cultured separately from neonatal rat brain were compared. Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed that whereas no cell type-specific differences in initial mtDNA damage were detected, mtDNA repair in oligodendrocytes, oligodendrocyte progenitors, and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, >60% of N-methylpurines were removed from the mtDNA by 24 hr. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes, oligodendrocyte progenitors, and microglia during the same time period. Mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis. In the present study, apoptosis, as determined by DNA laddering and ultrastructural analysis, was clearly induced by MNU treatment of cultured oligodendrocyte progenitors and microglia, but not in astroglia. These data demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis. However, further experimentation is necessary to determine if a causal relationship exists and contributes to the vulnerability of oligodendroglia following exposure to N-nitroso compounds in the environment or in chemotherapeutic regimen.

Multiple lines of evidence suggest involvement of oxidative stress in the pathogenesis of Alzheimer disease (AD). The finding that amyloid beta peptide (A beta) has neurotoxic properties and that such effects are mediated in part by free-radicals has provided an avenue to explore new therapeutic strategies. In this study, we showed that exposure of PC 12 cells to an A beta fragment induces oxidative damage of mitochondrial DNA. Cells were exposed for 24 h to 50 microM A beta (25-35) or to 50 microM of a control peptide with a scrambled sequence. Oxidative damage of mitochondrial DNA (mtDNA) was assessed using a Southern blot technique and an mtDNA-specific probe recognizing a 13.5-kilobase restriction fragment. Treatment of DNA with NaOH was used to reveal abasic sites and single strand breaks. Treatment with endonuclease III or FAPy glycosylase was used to detect pyrimidine or purine lesions, respectively. Cells exposed to A beta exhibited marked oxidative damage of mtDNA as evidenced by characteristic changes on Southern blots. Cells exposed to the scrambled peptide did not show such modifications. Simultaneous addition of the pineal hormone melatonin consistently prevented the A beta-induced oxidative damage to mtDNA. Mitochondrial dysfunction in AD has been demonstrated by several laboratories. This study provides experimental evidence supporting a causative role of A beta in mitochondrial lesions of AD.

Calcium receptor proteins are an essential link between hormones that alter intracellular calcium levels and the generation of cellular responses. However, there is no information available regarding the role of calcium receptor proteins, in particular the S100 family, in insulin action and/or diabetes. This study examines the effects of streptozotocin-induced type I diabetes on the expression of the individual S100A1 and S100B isoforms as well as their binding proteins. Diabetes did not increase (or initiate) S100B expression in any non-S100B-expressing tissue (skeletal muscle, heart, kidney, liver, spleen, and pancreas). In all S100B-expressing tissues examined (brain, white fat, and testes), S100B protein levels increased approximately 2-fold while steady state S100B messenger RNA (mRNA) levels decreased. S100A1-expressing tissues exhibited increased (kidney and lung), decreased (skeletal muscle), and unchanged (brain and heart) S100A1 protein levels. While noncoordinate changes in S100A1 protein and steady state mRNA levels were observed in heart, other S100A1-expressing tissues (brain, slow twitch skeletal muscle, and kidney) exhibited coordinate changes in S100A1 protein and steady state mRNA levels. Altogether, these results suggest that the effects of diabetes on S100 expression are isoform as well as tissue-specific. Gel overlay analysis of the S100-binding protein profile revealed both increases and decreases in binding proteins in all tissues examined. In summary, changes in the expression of S100A1, S100B, and S100-binding proteins occur in type I diabetes and represent important molecular events in the effects of insulin/insulin insufficiency on cell function.

CD20 is a B-lineage-specific gene expressed at the pre-B-cell stage of B-cell development that disappears on differentiation to plasma cells. As such, it serves as an excellent paradigm for the study of lineage and developmental stage-specific gene expression. Using in vivo footprinting we identified two sites in the promoter at -45 and -160 that were occupied only in CD20+ B cells. The -45 site is an E box that binds basic helix-loop-helix-zipper proteins whereas the -160 site is a composite PU.1 and Pip binding site. Transfection studies with reporter constructs and various expression vectors verified the importance of these sites. The composite PU.1 and Pip site likely accounts for both lineage and stage-specific expression of CD20 whereas the CD20 E box binding proteins enhance overall promoter activity and may link the promoter to a distant enhancer.

Genomic DNA is constantly being damaged and repaired and our genomes exist at lesion equilibrium for damage created by endogenous mutagens. Mitochondrial DNA (mtDNA) has the highest lesion equilibrium frequency recorded; presumably due to damage by H2O2 and free radicals generated during oxidative phosphorylation processes. We measured the frequencies of single strand breaks and oxidative base damage in mtDNA by ligation-mediated PCR and a quantitative Southern blot technique coupled with digestion by the enzymes endonuclease III and formamidopyrimidine DNA glycosylase. Addition of 5 mM alloxan to cultured rat cells increased the rate of oxidative base damage and, by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the single strand breaks and oxidative base damage frequency decreased to levels slightly below normal at 4 h and returned to normal levels at 8 h, the overshoot at 4 h being attributed to an adaptive up-regulation of mitochondrial excision repair activity. Guanine positions showed the highest endogenous lesion frequencies and were the most responsive positions to alloxan-induced oxidative stress. Although specific bases were consistently hot spots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner. The data reveal non-random oxidative damage to several nucleotides in mtDNA and an apparent adaptive, non-strand selective response for removal of such damage. These are the first studies to characterize oxidative damage and its subsequent removal at the nucleotide level in mtDNA.

CD19 expression begins at the pro-B cell stage of B cell development. As such it serves as a good prototype for B cell-specific genes whose expression begins shortly after lineage commitment. To understand the molecular mechanisms controlling CD19 gene expression, we isolated and functionally characterized the CD19 promoter using in vivo footprinting, gel shift assays, and transfection studies. Reporter constructs spanning portions of the promoter identified a region between -85 and -200 that produced high levels of reporter gene activity in lymphoid cells. In vivo footprinting identified protected regions over the known high affinity B cell lineage-specific activator protein (BSAP) site, the low affinity BSAP site, a SP1/Egr-1 site termed the CD19 GC box, and two novel sites named the AT box and PyG box. Phorbol ester treatment of a pre-B cell line up-regulated CD19 expression, induced Egr-1, and enhanced the footprint over the GC box. Gel shift assays demonstrated SP1 and Egr-1 binding to the CD19 GC box, while unknown nuclear proteins bound the PyG and AT boxes. Mutations in the AT box or in the BSAP sites did not affect CD19 reporter construct activity, while a mutation of the GC box reduced it modestly, and a PyG box mutation reduced it dramatically. BSAP failed to trans-activate CD19 promoter constructs in B cells or non-B cells, suggesting that cis elements such as the PyG and GC boxes are also necessary for high level CD19 promoter expression.

Increasing evidence indicates that nitric oxide (NO) may play a role in immune-mediated injury to beta-cells. One site for the action of this agent is the mitochondrion. Although the exact targets for damage within this organelle have yet to be fully elucidated, a potential location for injury is mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by NO. Both exogenous NO generation (spermine/NO adduct [sper/NO]) and endogenous production of NO (IL-1beta) were studied. To study the effects of exogenously produced NO, neonatal rat islet cells in monolayers were exposed to varying doses of sper/NO for 30 min. Total cellular DNA was isolated and treated with alkali to produce strand breaks at abasic sites resulting from exposure to NO. Damage to mtDNA was evaluated using a quantitative Southern blot technique. The results showed that sper/NO caused dose-dependent damage to mtDNA. Additionally, mtDNA was found to be more sensitive to injury generated by either source than a similarly sized fragment of nuclear DNA. To evaluate the effects of endogenously produced NO, beta-cell cultures were treated with IL-1beta for 18 h. Other cultures were treated with IL-1beta and an inhibitor of the inducible form of nitric oxide synthase, aminoguanidine. DNA was evaluated as described for the sper/NO studies. IL-1beta caused appreciable damage to mtDNA, and this damage was reduced in mtDNA from cultures treated with IL-1beta and aminoguanidine. These studies show that mtDNA is a sensitive target for NO generated both endogenously and exogenously and that this DNA is more vulnerable to NO-induced damage than nuclear DNA.

This study was designed to investigate the repair of oxidative damage in nuclear DNA sequences with different transcriptional activities. Chinese hamster ovary (CHO) cells were treated with the oxygen radical generator hypoxanthine/xanthine oxidase (Hyp/XO). Damage and repair were evaluated in 14-kb restriction fragments containing either the DHFR gene, a 3'-non-transcribed flanking region, or the c-fos gene using a quantitative Southern blot technique. Damage to the sugar-phosphate backbone and abasic sites were detected by measuring their lability in alkali conditions. Lesions in DNA bases were identified using the bacterial repair enzyme endonuclease III, which predominantly recognizes damage to thymines and cytosines, and formamidopyrimidine-DNA glycosylase, which recognizes 8-oxoguanine and purines with fractured imidazole rings. The results showed that similar amounts of all types of oxidative damage were produced in both the transcribed and non-transcribed sequences following a 1-h exposure to the radical generator. Repair in all sequences was rapid, with approximately 60% removal of lesions observed by 1 h. Therefore, within these sequences, the repair of oxidative lesions is much faster than that of other types of damage, such as those induced by alkylating toxins and UV irradiation, and the repair is not affected appreciably by transcriptional status.

Normal and malignant cells of the oligodendrocyte lineage show increased sensitivity to alkylating agents compared to astrocytes. One of the most mutagenic DNA lesions formed following exposure to alkylating agents is O6-alkylguanine. To determine whether the increased sensitivity to nitrosoureas seen in oligodendrocytes is due to decreased repair capacity for O6-alkylguanine, removal of this lesion from DNA was assessed in primary cultures of rat oligodendrocytes, astrocytes, and microglia. Glial cells were exposed to 1 mM N-methyl-N-nitrosourea for 1 h and allowed 8 or 24 h for repair. Repair was evaluated using an immunoslot blot technique and a monoclonal antibody which recognizes O6-methylguanine (O6MeGua). Astrocytes removed O6MeGua more efficiently (approximately 80% in 24 h) than either oligodendrocytes (approximately 20%) or microglia (approximately 4%). Determination of O6-alkylguanine-DNA-alkyltransferase (AT) activity revealed that astrocytes contain 0.4 pmol/mg protein, which is average by comparison to other cell types. Both oligodendrocytes and microglia exhibited very low levels of AT (oligodendrocytes, 0.08; microglia, 0.01 pmol/mg protein). These data are the first to show that within different populations of glial cells, O6MeGua adduct removal is substantially reduced in both oligodendrocytes and microglia. Rapid removal of O6MeGua in astrocytes coupled with persistence of this mutagenic lesion in oligodendrocytes following exposure of the developing central nervous system to nitrosoureas could contribute to the observed formation of oligodendrogliomas. Inefficient removal of O6MeGua in oligodendrogliomas might also account for their response to chemotherapeutic regimens involving alkylating agents such as procarbazine, lomustine, and carmustine. The lack of repair of O6MeGua in microglia suggests that primary lymphomas of the central nervous system might be sensitive to treatment with alkylating drugs whose toxicity depends on repair of this adduct.

The role of poly(ADP-ribose) polymerase (PADPRP) in nuclear DNA repair and other nuclear processes has been intensely studied and debated for decades. Recent studies have begun to shed new light on these arguments with firm experimental data for its role, primarily, as a remodeler of chromatin structure. Those studies imply that PADPRP plays an indirect role in DNA repair, serving to expose DNA to repair enzymes through chromatin remodeling. Only DNA that is tightly packaged would require PADPRP activity for its repair; while DNA in an open conformation would be accessible to DNA repair enzymes and not require PADPRP activity. The purpose of the current studies was to address the above hypothesis directly. Using quantitative Southern blot analysis, we studied repair in transcribed and nontranscribed nuclear DNA sequences in ADPRT 351 cells 95% deficient in PADPRP activity. Cells were exposed to methylnitrosourea (MNU) for 1 h and allowed to repair for 8 or 24 h. Densitometric scans of autoradiographs revealed that, when compared to their parental V79 cell line, ADPRT 351 cells 95% deficient in PADPRP activity were equally as efficient in repair of N-methylpurines in the transcribed sequence containing the dihydrofolate reductase gene. However, the ADPRT 351 cells were deficient in the ability to repair these lesions in the nontranscribed sequence containing the IgE gene compared to repair of the same sequence in the parental V79 cells. Nucleoid sedimentation assays demonstrated that the ADPRT 351 cells are deficient in repair across the entire genome when compared to the parental V79 cells. These studies indicate that PADPRP activity is not required for repair of N-methylpurines in transcribed nuclear DNA sequences but is necessary for the repair of these lesions in nontranscribed nuclear DNA sequences as well as across the entire genome since the DNA in a given cell is predominantly nontranscribed.

Recent evidence has linked mitochondrial DNA (mtDNA) damage to several disease processes,including cancer and aging. An important source of such damage is reactive oxygen species. These molecules can be generated endogenously via the electron transport system or may arise from a host of exogenous sources. It has been reported that extracts from cells of individuals with xeroderma pigmentosum group A (XP-A) do not repair some types of oxidative DNA damage. The current experiments were designed to determine whether there is a correlation between the inadequate repair of oxidatively damaged nuclear DNA in XP-A cells and the capacity of such cells to repair similar damage to their mtDNA. The ability of karyotypically normal human fibroblasts (WI-38) and XP-A fibroblasts to repair alloxan-generated oxidative damage to nuclear and mtDNA was assessed using a quantitative Southern blot method in conjunction with the repair enzymes endonuclease III and formamidopyrimidine DNA glycosylase. The data indicate that both nuclear and mtDNA repair of each damage type investigated is more efficient in the WI-38 cells. These findings suggest a similarity between the process(es) used to repair oxidative damage to nuclear and mtDNA in that both are inhibited by the defect in XP-A.

Damage to mitochondrial DNA (mtDNA) has recently been associated with a variety of human diseases including cancer, diabetes mellitus, and aging. The mechanisms by which the mitochondria respond to DNA damage are of prime importance in understanding how damage can persist and cause disease. Here we demonstrate the repair of mitochondrial DNA damage induced by the naturally occurring, radiomimetic drug bleomycin. WI-38 cells were first permeabilized using 20 micrograms/ml lysophosphatidylcholine in order to increase the intracellular concentration of bleomycin. Dose response studies with the permeabilized cells showed that a concentration of 5 micrograms/ml bleomycin given for 30 min caused sufficient DNA damage for repair studies. Following treatment with this concentration of bleomycin, repair of mtDNA damage was found to be about 80% by 2 h. However, after 4 h no additional repair was observed. The results indicate that there is an efficient DNA repair system in human mitochondria for some types of damage caused by bleomycin. However, there is a component of damage caused by this agent that either is not repaired or is removed at a much slower rate.

The CD19, CD20 and CD22 genes encode transmembrane proteins that are of vital importance to B-cell function. Similar to the immunoglobulin (Ig) genes, they are expressed in a lineage-specific and developmentally regulated manner. Here, John Kehrl and colleagues describe how an understanding of the transcriptional regulation of the CD19, CD20 and CD22 genes is leading to valuable insights into some of the important molecular events that occur in B-cell development and differentiation.

A growing body of evidence suggests that a variety of chronic diseases, including cancer and diabetes, are associated with damage to mitochondrial DNA. Since mitochondria are constantly exposed to high levels of reactive oxygen species, it is likely that oxidative damage to mitochondrial DNA may be responsible for some of these maladies. To determine whether mitochondria can repair this damage, a quantitative Southern blot technique was utilized to identify repair in specific DNA fragments. A 10.8-kilobase mitochondrial restriction fragment was studied employing a probe containing the entire mouse mitochondrial genome. Alloxan was employed to generate oxygen radicals. Insulinoma cells were exposed to alloxan for 1 h, and total cellular DNA was isolated immediately or after intervals of up to 8 h. Alkali treatment was used to identify abasic sites and sugar lesions, endonuclease III was used to identify lesions associated with thymine and cytosine damage, and formamidopyrimidine-DNA glycosylase was employed to recognize formamidopyrimidines and 8-oxoguanines in DNA. The results showed that all forms of damage studied were repaired by 4 h, indicating that mitochondria are able to efficiently repair damage to their DNA caused by reactive oxygen species.

Because of the possible involvement of O6-methyldeoxyguanosine as a cytotoxic and carcinogenic lesion in pancreatic beta-cells, studies were undertaken to assess the ability of rat beta-cells to repair this DNA lesion. Primary cultures of neonatal rat beta-cells were shown to contain very low levels of O6-methylguanine-DNA-methyltransferase activity, the predominant mechanism for repairing O6-methyldeoxyguanosine in mammalian cells. However, using a 32P-endlabeling assay to measure O6-methyldeoxyguanosine in cells after exposure to N-methyl-N-nitrosourea, it was determined that rat beta-cells repaired O6-methyldeoxyguanosine to a substantial extent over a 24-h period. To elucidate the mechanism of O6-methyldeoxyguanosine repair in the virtual absence of constitutive O6-methylguanine-DNA-methyltransferase expression, studies were performed to determine if O6-methylguanine-DNA-methyltransferase expression was enhanced in N-methyl-N-nitrosourea-treated beta-cells. No increase in O6-methylguanine-DNA-methyltransferase activity was detected 24 or 48 h after exposure. However, Northern blot analysis showed a two- to threefold elevation in O6-methylguanine-DNA-methyltransferase messenger RNA levels in beta-cells 12 and 24 h after N-methyl-N-nitrosourea treatment. This finding is the first demonstration of a change in O6-methylguanine-DNA-methyltransferase messenger RNA levels in a cell type with low constitutive activity.

Extrapancreatic activity of the sulfonylurea, glipizide, was evaluated in the neonatal streptozotocin-induced rat model of noninsulin-dependent diabetes. Two day old Wistar rats were given a bolus of streptozotocin (90 mg/kg i.p.) to cause noninsulin-dependent diabetes; these animals became severely glucose intolerant and eventually developed a cardiomyopathy characterized by reduced heart rate, contractility and cardiac output. Male littermates injected with citrate buffer served as nondiabetic controls. At four weeks of age, the nondiabetic and NIDD rats were administered by gavage either glipizide (2.5 mg/kg) or the methyl cellulose vehicle. Throughout the treatment protocol, no difference in the degree of glucose intolerance was observed between the glipizide-treated and vehicle-treated animals. Glipizide therapy also was ineffective in improving plasma insulin levels, which were significantly depressed in the diabetic group. Yet, animals treated with glipizide for one year exhibited improved myocardial contractile function relative to the vehicle-fed or ad lib fed diabetic animals. Heart rate was significantly elevated and there was a tendency for both the rate of relaxation and contractility to be elevated in sulfonylurea-treated group. Glipizide also reduced the degree of insulin resistance in the heart. Since these changes occur in the absence of changes in glucose tolerance or insulin levels, the heart appears to be very sensitive to the direct effects of the sulfonylureas.

CD22 is a B lineage-restricted member of the Ig superfamily that serves as an adhesion receptor expressed by mature B lymphocytes. In this study, the ability of different cell types to attach to COS cells transiently transfected with a full-length CD22 cDNA (COS-CD22) was examined to determine the cellular distribution of the ligand for CD22. T and B lymphocytes, monocytes, erythrocytes, and neutrophils formed specific rosettes with COS-CD22 cells at 4 degrees C. A panel of 33 new mAb directed against CD22 were developed to examine the regions of CD22 that mediate adhesion. Four of these mAb, HB22-7, -22, -23, and -33 (at 1 to 5 micrograms/ml) specifically blocked adhesion (75 to 95%) of all cell types to COS-CD22 cells. Each of these mAb cross-blocked each other's binding, suggesting that ligand binding occurs through a single region of CD22. These mAb also identify a region of CD22 distinct from those defined by previously described CD22 mAb. CD22-mediated adhesion of cell lines to COS-CD22 cells was independent of CD45RO and CDw75 expression, and it was not inhibited by mAb against known integrins. Although alpha-2,6-linked sialic acid expressed on the surface of COS cells did not serve as a ligand for CD22, the CD22 ligand may contain a critical sialic acid determinant, as neuraminidase treatment of all target cells eliminated CD22-mediated adhesion. CD22-mediated adhesion was Ca2+/Mg2+ independent, again suggesting that integrins were not involved. An inhibitory substance for CD22-mediated adhesion was found to be present in FCS and some ascites fluid. Analysis of CD22 mRNA and protein revealed that although multiple mRNA splice variants of CD22 mRNA can be detected, only a single protein isoform was detected on the cell surface. Therefore, although the identity of the CD22 ligands remains incompletely characterized, it is possible that a single major ligand is expressed by RBC and leukocytes, which binds to a single region of CD22.

The human CD22 gene is expressed specifically in B lymphocytes and likely has an important function in cell-cell interactions. A nearly full length human CD22 cDNA clone was used to isolate genomic clones that span the CD22 gene. The CD22 gene is spread over 22 kb of DNA and is composed of 15 exons. The first exon contains the major transcriptional start sites. The translation initiation codon is located in exon 3, which also encodes a portion of the signal peptide. Exons 4 to 10 encode the seven Ig domains of CD22, exon 11 encodes the transmembrane domain, exons 12 to 15 encode the intracytoplasmic domain of CD22, and exon 15 also contains the 3' untranslated region. A minor form of CD22 mRNA likely results from splicing of exon 5 to exon 8, skipping exons 6 and 7. A 4.6-kb XbaI fragment of the CD22 gene was used to map the chromosomal location of CD22 by fluorescence in situ hybridization. The hybridization locus was identified by combining fluorescent images of the probe with the chromosomal banding pattern generated by an Alu probe. The results demonstrate that CD22 is located within the band region q13.1 of chromosome 19. Two closely clustered major transcription start sites and several minor start sites were mapped by primer extension. Similarly to many other lymphoid-specific genes, the CD22 promoter lacks an obvious TATA box. Approximately 4 kb of DNA 5' of the transcription start sites were sequenced and found to contain multiple Alu elements. Potential binding sites for the transcriptional factors NF-kappa B, AP-1, and Oct-2 are located within 300 bp 5' of the major transcription start sites. A 400-bp fragment (bp -339 through +71) of the CD22 promoter region was subcloned into a pGEM-chloramphenicol acetyltransferase vector and after transfection into B and T cells was found to be active in both B and T cells. Further studies of the CD22 gene should lead to a greater understanding of the expression of CD22 during B cell development and differentiation.

Using subtractive cDNA cloning, we have isolated a series of cDNA clones that are differentially expressed between B and T lymphocytes. Whereas some of the isolated cDNA are from known B cell-specific genes, many of them represent previously uncharacterized genes. One of these unknown genes was denoted as BL34. Northern blot analysis performed with the BL34 cDNA revealed a 1.6-kb mRNA transcript that was present at low levels in RNA extracted from resting B lymphocytes, but whose expression was markedly increased in RNA prepared from mitogen-activated B cells. Similarly, RNA prepared from several B cell lines treated with phorbol myristate acetate (PMA) contained high levels of BL34 mRNA. In contrast, RNA from purified T cells treated with phytohemagglutinin and PMA had undetectable amounts of BL34 mRNA. In addition, high levels of BL34 mRNA were detected in RNA purified from PBMC of a patient with B cell acute lymphocytic leukemia. Southern blot analysis of human DNA from various tissues and cells lines demonstrated that BL34 is a single-copy gene without evidence of rearrangement. Two full length BL34 cDNA were sequenced, and an open reading frame of 588 bp was identified that was predicted to encode for a 196 amino acid protein. Searches of several protein data bases failed to find any homologous proteins. To directly analyze the expression of BL34 mRNA in lymphoid tissues in situ, hybridization studies with human tonsil tissue sections were performed. BL34 mRNA was detected in a portion of the cells in the germinal center region and adjacent to the mantle region. Further characterization of the BL34 gene and its protein should lead to insights to its role in B cell function and the consequences of its over-expression in acute lymphocytic leukemia.

Previous work from our laboratory has shown that mitochondria are able to repair N-methylpurines formed by methylnitrosourea (MNU). However, it is unclear as to whether repair mechanisms that remove this type of lesion in nuclear DNA also remove these adducts in mitochondria. To address this question, we studied repair of MNU-induced N-methylpurines in the mitochondrial DNA from xeroderma pigmentosum complementation group D (XP-D) cells using quantitative Southern blot analysis and 32P-end-labeling techniques. These cells have been reported to be defective in the repair of this type of lesion in their nuclear genome. WI 38 cells were used as normal controls for these studies. Both XP-D fibroblasts and WI 38 cells were exposed to 0.5 mM MNU for 1 h. Following an 8 h repair period, 61% of N-methylpurines were repaired in the mitochondrial genome of XP-D cells and 39% of these lesions were repaired in WI 38 cells. After 24 h, XP-D cells had repaired 77% of the N-methylpurines in their mitochondrial genome, while WI 38 cells had 44% repair of this type of damage. During this same 24 h time period, 81.5% of the N7-methylguanines had been removed from the total cellular DNA of the WI 38 cells compared to only 38.3% repair of this lesion in the XP-D cells. Thus, XP-D cells, though deficient in the repair of N-methylpurines in their nuclear genome, are proficient in the repair of this type of damage in their mitochondria, suggesting that the mechanisms to repair N-methylpurines in the nuclear and mitochondrial genomes of these cells are different.