An immortalized in vitro model of the blood-brain barrier: glutathione levels and sensitivity to oxidative stress.
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Animals↗
Biomedical subjects
J B Clark
Publications and source records attributed to J B Clark.
Pretreatment of astrocytes with interferon-alpha/beta prevents neuronal mitochondrial respiratory chain damage.
Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Interferon-beta is used for the treatment of multiple sclerosis, but currently little is known regarding its mode of action. Exposure of astrocytes in culture to interferon-gamma plus lipopolysaccharide results in stimulation of nitric oxide release. Using a coculture system, we have been able to use astrocytes as a source of nitric oxide/peroxynitrite in an attempt to "model" the effects of raised cytokine levels observed in multiple sclerosis and to monitor the effect on neurones. Our results indicate that stimulation of astrocytic nitric oxide synthase activity causes significant damage to the mitochondrial activities of complexes II/III and IV of neighbouring neurones. This damage was prevented by a nitric oxide synthase inhibitor, suggesting that the damage was nitric oxide-mediated. Furthermore, interferon-alpha/beta also prevented this damage. In view of these results, we suggest that a possible mechanism of action of interferon-beta in the treatment of multiple sclerosis is that it prevents astrocytic nitric oxide production, thereby limiting damage to neighbouring cells, such as neurones.
Animals↗
Distribution of mRNAs encoding the peroxisome proliferator-activated receptor alpha, beta, and gamma and the retinoid X receptor alpha, beta, and gamma in rat central nervous system.
We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPAR alpha, PPAR beta, and PPAR gamma and the rat retinoid X receptor (RXR) isoforms RXR alpha, RXR beta, and RXR gamma. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are ubiquitously present in different brain regions during development, PPAR gamma mRNA is essentially undetectable, and RXR gamma mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are present in all cell types, albeit that PPAR alpha and RXR alpha mRNAs are at levels near the limit of detection in CGNs. PPAR gamma mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPAR alpha mRNA in adult astrocytes. RXR gamma mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.
Animals↗
3-Hydroxybutyrate aids the recovery of the energy state from aglycaemic hypoxia of adult but not neonatal rat brain slices.
The level of phosphocreatine (PCr) and the intracellular pH (pHi) of superfused cortical brain slices from adult or 10-day-old rats were monitored using 31P NMR. When the glucose in the superfusing medium was replaced by 3-hydroxybutyrate (3HB), there was a significant reduction in PCr of the adult but not the neonatal slices. The level of PCr of the adult slices was reduced by a greater amount by aglycaemic hypoxia compared with the neonatal brain slices and pHi was decreased by the same amount. After aglycaemic hypoxia, the levels of PCr of the neonatal slices recovered to the same extent when perfused with glucose or 3HB alone or a mixture of glucose and 3HB. The recovery of the PCr was significantly more in the neonatal than the adult brain slices with glucose alone after aglycaemic hypoxia, whereas pHi returned to control levels in both tissue types and with all substrates. The relative recovery of the PCr of the adult slices after aglycaemic hypoxia was the same with either 3HB or glucose. However, if glucose and 3HB were applied together, recovery of PCr was significantly improved compared with glucose alone.
3-Hydroxybutyric Acid↗
Peroxynitrite and brain mitochondria: evidence for increased proton leak.
Peroxynitrite has been reported to inhibit irreversibly mitochondrial respiration. Here we show that three sequential additions of 200 microM peroxynitrite (initial concentration) to rat brain mitochondria (0.2 mg of protein/ml) significantly stimulated state 4 respiration and that further additions progressively inhibited it. No stimulation of state 3 respiration or of the maximal enzymatic activities of the respiratory chain complexes was observed on identical peroxynitrite exposure. State 4 respiration is a consequence of the proton permeability of the mitochondrial inner membrane, and we demonstrate that the peroxynitrite-induced stimulation of state 4 respiration is accompanied by a decreased mitochondrial membrane potential, suggesting an increase in this proton leak. Cyclosporin A did not affect the stimulation, suggesting no involvement of the mitochondrial permeability transition pore. The stimulation was prevented by the lipid-soluble vitamin E analogue Trolox, suggesting the involvement of lipid peroxidation, a proposed mechanism of peroxynitrite cytotoxicity. Lipid peroxidation has previously been reported to increase membrane bilayer proton permeability. The high polyunsaturate content of brain mitochondrial phospholipids may predispose them to peroxidation, and thus a peroxynitrite-induced, lipid peroxidation-mediated increase in proton leak may apply particularly to brain mitochondria and to certain neurodegenerative disorders thought to proceed via mechanisms of mitochondrial oxidative damage.
Animals↗
Hormonal regulation of the mRNA encoding the ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in neonatal primary cultures of cortical astrocytes and meningeal fibroblasts.
We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3-hydroxy-3-methylglutaryl-CoA cycle, namely, mitochondrial acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum-free/hormone-free medium.
Animals↗
Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences.
Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were determined from nine species in the Drosophila saltans species group. The partition homogeneity test and partitioned Bremer support were used to measure incongruence between phylogenetic hypotheses generated from individual partitions. Individual loci were generally congruent with each other and consistent with the previously proposed morphological hypothesis, although they differed in level of resolution. Since extreme conflict between partitions did not exist, the data were combined and analyzed simultaneously. The total evidence method gave a more resolved and highly supported phylogeny, as indicated by bootstrap proportions and decay indices, than did any of the individual analyses. The cordata and elliptica subgroups, considered to have diverged early in the history of the D. saltans group, were sister taxa to the remainder of the saltans group. The sturtevanti subgroup, represented by D. milleri and D. sturtevanti, occupies an intermediate position in this phylogeny. The saltans and parasaltans subgroups are sister clades and occupy the most recently derived portion of the phylogeny. As with previous morphological studies, phylogenetic relationships within the saltans subgroup were not satisfactorily resolved by the molecular data.
Alcohol Dehydrogenase↗
Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group.
Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.
Animals↗
N-acetyl aspartate: a marker for neuronal loss or mitochondrial dysfunction.
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Animals↗
Unintended pregnancy among female soldiers presenting for prenatal care at Madigan Army Medical Center.
More than half of all pregnancies in this country are unintended at the time of conception. Although women make up 13% of the U.S. Army, there are no epidemiological studies of unintended pregnancy in U.S. Army soldiers. This descriptive study was conducted to determine the prevalence of and factors associated with unintended pregnancy in female soldiers presenting for prenatal care at Madigan Army Medical Center. All soldiers presenting for prenatal care from mid-February 1996 through mid-February 1997 were asked to fill out an anonymous, 12-page written questionnaire. All but one soldier (99.7%) agreed to participate (N = 347). Overall, 55% of soldiers presenting for prenatal care reported that their pregnancies were unintended at the time of conception. The majority of officers (60%) and noncommissioned officers (65%) reported that their pregnancies were intended. In contrast, only 39% of the junior enlisted soldiers reported that their pregnancies were intended at the time of conception. The majority of women (62%) who reported that their pregnancies were unintended were not using any form of birth control during the month they conceived. The most common reason for not using birth control was the stopping of contraception because of side effects. The most common contraceptive method used by those women who reported birth control use during the month they conceived was the male condom. The majority of pregnancies among junior enlisted soldiers presenting for prenatal care at Madigan Army Medical Center are unintended. Prevention programs should target this subpopulation of soldiers and address knowledge, attitudes, and beliefs related to the use of contraception among those at risk for unintended pregnancy.
Adult↗
A phylogenetic perspective on P transposable element evolution in Drosophila.
The P element, originally described in Drosophila melanogaster, is one of the best-studied eukaryotic transposable elements. In an attempt to understand the evolutionary dynamics of the P element family, an extensive phylogenetic analysis of 239 partial P element sequences has been completed. These sequences were obtained from 40 species in the Drosophila subgenus Sophophora. The phylogeny of the P element family is examined in the context of a phylogeny of the species in which these elements are found. An interesting feature of many of the species examined is the coexistence in the same genome of P sequences belonging to two or more divergent subfamilies. In general, P elements in Drosophila have been transmitted vertically from generation to generation over evolutionary time. However, four unequivocal cases of horizontal transfer, in which the element was transferred between species, have been identified. In addition, the P element phylogeny is best explained in numerous instances by horizontal transfer at various times in the past. These observations suggest that, as with some other transposable elements, horizontal transfer may play an important role in the maintenance of P elements in natural populations.
Animals↗
Stimulation of glyceraldehyde-3-phosphate dehydrogenase by oxyhemoglobin.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme regulated by many diverse mechanisms. In this study we present evidence that GAPDH activity is stimulated in the presence of oxyhemoglobin (2.3-fold, P < 0.005). No stimulation was seen by myoglobin, and only slight stimulation (1.2-fold, not significant) by methemoglobin was observed. Such stimulation may have physiological significance as 1,3-bis-phosphoglycerate, the product of GAPDH, isomerises to 2,3-bis-phosphoglycerate, an allosteric effector that decreases the oxygen affinity of hemoglobin, thus providing a feedback loop. The results suggest that when assaying GAPDH activity in biological samples, hemoglobin content should be taken into account.
Animals↗
Pertussis: an update on primary prevention and outbreak control.
Pertussis is a highly contagious respiratory disease. Infected adolescents and adults with mild illness are the source of potentially life-threatening illness in infants and young children. The incidence of pertussis has been rising steadily in recent years. Primary vaccination is 80 percent effective, but protection is transient. Pertussis can be difficult to diagnose because classic whooping cough is uncommon, disease manifestations are often atypical, and laboratory and radiologic aids are frequently nonspecific. Diagnosis is usually based on the clinical picture, but culture, direct fluorescent antibody tests and serology can be helpful. Antibiotic therapy can decrease the duration and severity of illness, and prevents secondary spread if started early. Effective management of pertussis outbreaks requires early diagnosis and treatment of cases, antibiotic prophylaxis of contacts and accelerated vaccination of susceptible infants and children. Acellular pertussis vaccine preparations have recently been recommended for the entire primary vaccination series.
Adolescent↗
[U-13C]glutamate metabolism in rat brain mitochondria reveals malic enzyme activity.
13C nuclear magnetic resonance spectroscopy was used to study the activity of malic enzyme in isolated brain mitochondria from rat in the presence of unlabelled malate and [U-13C]glutamate. ADP, inorganic phosphate, malate and [U-13C]glutamate were added to a suspension of oxygenated mitochondria. Typical tricarboxylic acid (TCA) cycle constituents (malate, 2-oxoglutarate and succinate) were labelled from [U-13C]glutamate and detected in the superfusion medium. The labelling patterns in the different atom positions of glutamate revealed entry of both unlabelled and labelled acetyl-CoA into the TCA cycle. Unlabelled acetyl-CoA was derived via pyruvate from exogenously applied malate by the action of mitochondrial malic enzyme, while labelled acetyl-CoA was derived from TCA cycle intermediates, most likely by the action of mitochondrial malic enzyme on malate produced from [U-13C]glutamate. The results demonstrate malic enzyme activity and pyruvate recycling in isolated rat brain mitochondria.
Animals↗
Nitric oxide-induced blood-brain barrier dysfunction is not mediated by inhibition of mitochondrial respiratory chain activity and/or energy depletion.
Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the breakdown of blood-brain barrier (BBB) function which can occur during various inflammatory conditions. Recent evidence suggests a role for the free radical nitric oxide (NO) in the process of cytokine-induced barrier dysfunction. The mitochondrial enzyme cytochrome oxidase is inhibited by NO, and hence, using a coculture model of the BBB, we have investigated whether TNF-alpha alters barrier function by a NO-mediated mechanism and, if so, whether it is related to a reduction of endothelial cell respiration and ATP synthesis. TNF-alpha mediated a marked reduction in model BBB integrity that was partially prevented by inhibition of NO synthase activity. Additionally, exposure of BBB cultures to authentic gaseous NO also resulted in a progressive decline in barrier integrity. Authentic NO inhibited endothelial cell respiration in a reversible manner. Mitochondrial respiratory chain inhibitors induced significant reductions in endothelial cell respiratory rate and ATP levels, but did not mimic the action of NO on barrier function. We conclude that NO is partially responsible for the detrimental effect of TNF-alpha on BBB function. The mechanism of NO-induced barrier dysfunction does not involve an inhibition of endothelial mitochondrial electron transport chain and reduced energy resources.
Animals↗
Raised serum nitrate and nitrite levels in patients with multiple sclerosis.
Nitric oxide and its highly reactive derivative peroxynitrite have been implicated as non-specific inflammatory mediators of neuronal and oligodendrocyte damage and death in multiple sclerosis. In a cross-sectional study we found levels of the nitric oxide metabolites nitrate and nitrite to be raised in the serum of patients with demyelinating disease (65.6 microM (SD 32.9)), acquired immune deficiency syndrome (57.9 microM (SD 34.9)) and inflammatory neurological disease (57.5 microM (SD 31.3)), compared with normal control subjects (32.8 microM (SD 12.2)) and patients with non-inflammatory neurological disease (41.1 microM (SD 12.3), p < 0.001). Nitric oxide metabolites were raised in all clinical subtypes of multiple sclerosis, as well as in clinically isolated syndromes compatible with demyelination, and were not related to progressive disease or disability. This study provides further evidence for a role of nitric oxide in the immunopathogenesis of inflammatory diseases of the central nervous system, including multiple sclerosis.
Acquired Immunodeficiency Syndrome↗
Interrelationships between astrocyte function, oxidative stress and antioxidant status within the central nervous system.
Astrocytes have, until recently, been thought of as the passive supporting elements of the central nervous system. However, recent developments suggest that these cells actually play a crucial and vital role in the overall physiology of the brain. Astrocytes selectively express a host of cell membrane and nuclear receptors that are responsive to various neuroactive compounds. In addition, the cell membrane has a number of important transporters for these compounds. Direct evidence for the selective co-expression of neurotransmitters, transporters on both neurons and astrocytes, provides additional evidence for metabolic compartmentation within the central nervous system. Oxidative stress as defined by the excessive production of free radicals can alter dramatically the function of the cell. The free radical nitric oxide has attracted a considerable amount of attention recently, due to its role as a physiological second messenger but also because of its neurotoxic potential when produced in excess. We provide, therefore, an in-depth discussion on how this free radical and its metabolites affect the intra and intercellular physiology of the astrocyte(s) and surrounding neurons. Finally, we look at the ways in which astrocytes can counteract the production of free radicals in general by using their antioxidant pathways. The glutathione antioxidant system will be the focus of attention, since astrocytes have an enormous capacity for, and efficiency built into this particular system.
Animals↗
Evaluation of the efficacy of potential therapeutic agents at protecting against nitric oxide synthase-mediated mitochondrial damage in activated astrocytes.
Within the central nervous system, nitric oxide is an important physiological messenger. However, when synthesized excessively in neurones, cell death may occur. An impairment of mitochondrial cytochrome oxidase and subsequent cellular energy depletion seems to be a likely mechanism for this neurotoxicity. Within neurones, nitric oxide is synthesized by the constitutive, Ca(2+)-dependent form of nitric oxide synthase (nNOS). Astrocytes, however, possess both the constitutive and the inducible Ca(2+)-independent NOS (iNOS), which is expressed by endotoxin and/or cytokines. In vitro, activation of nNOS rapidly produces neuronal cell death. In contrast to neurones, following induction of iNOS, astrocytes synthesize large quantities of nitric oxide, but cell death is not apparent despite marked damage to mitochondrial cytochrome oxidase. The resistance of astrocytes to nitric oxide synthase-mediated cell damage may be due to their ability to increase their glycolytic rate when mitochondrial ATP synthesis is compromised. On the basis of this phenomenon, we propose that activated astrocytes represent a suitable system for studying the efficacy of potential therapeutic agents at protecting from nitric oxide synthase-mediated mitochondrial damage.
Animals↗