Paroxysmal nocturnal hemoglobinuria: molecular pathogenesis and molecular therapeutic approaches

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic stem cell disorder classified as an intravascular hemolytic anemia. Abnormal blood cells are deficient in glycosylphosphatidyl inositol (GPI)-anchored proteins. Deficiencies of GPI-anchored complement regulatory proteins, such as decay accelerating factor (DAF) and CD59, render red cells very sensitive to complement and result in complement-mediated hemolysis and hemoglobinuria. In the affected hematopoietic cells from patients with PNH, the first step in biosynthesis of the GPI anchor is defective. Three genes are involved in this reaction step and one of them, an X-linked gene termed PIG-A, is mutated in affected cells. Granulocytes and lymphocytes from the same patient have the same mutation, indicating that a somatic PIG-A mutation occurs in hematopoietic stem cells. The PIG-A gene is mutated in all patients with PNH reported to date. We review these recent advances in the understanding of the molecular pathogenesis of PNH. Furthermore, we present an hypothesis regarding the predominance of the PNH clone, caused by positive selection by hematopoietic suppressive cytokines, such as transforming growth factor (TGF)-beta. In addition, we discuss the possibility of cure for PNH through molecular therapeutic strategy using gene transfer techniques. (Key words: paroxysmal nocturnal hemoglobinuria, glycosylphosphatidylinositol-anchored proteins, PIG-A, clonal dominance, growth advantage, transforming growth factor-beta, gene therapy, molecular therapeutic approach).     

Changes in mitochondrial function resulting from synaptic activity in the rat hippocampal slice

Digital imaging microfluorimetry was used to visualize changes in mitochondrial potential and intracellular Ca2+ concentration, [Ca2+]i, in thick slices of rat hippocampus. Electrical activity, especially stimulus train-induced bursting (STIB) activity, produced slow, prolonged changes in mitochondrial potential within hippocampal slices as revealed by fluorescence measurements with rhodamine dyes. Changes in mitochondrial potential showed both temporal and spatial correlations with the intensity of the electrical activity. Patterned changes in mitochondrial potential were observed to last from tens of seconds to minutes as the consequence of epileptiform discharges. STIB-associated elevations in [Ca2+]i were also prolonged and exhibited a spatial pattern similar to that of the mitochondrial depolarization. The mitochondrial depolarization was sensitive to TTX and glutamate receptor blockers ([Mg2+]o and CNQX or DNQX plus D-AP-5) and to the inhibition of glutamate release by activation of presynaptic NPY receptors. The monitoring of mitochondrial potential in slice preparations provides a new tool for mapping synaptic activity in the brain and for determining the roles of mitochondria in regulation of brain synaptic activity.     

Successful treatment of intractable epilepsia partialis continua with multiple subpial transections

Alpha hydroxy acids are used extensively by patients and consumers for restorative cutaneous purposes. The current study of 32 patients evaluated the clinical and psychosocial effects of this cosmetic therapy. After 12 weeks, significant clinical improvements were found for facial skin tone and fine wrinkling, as well as for patient-reported satisfaction with physical appearance and with marital or relationship quality.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.     

Postmarketing surveillance study of a non-chlorofluorocarbon inhaler according to the safety assessment of marketed medicines guidelines

OBJECTIVE: To evaluate the safety of a non-chlorofluorocarbon metered dose salbutamol inhaler. DESIGN: This was a postmarketing surveillance study, conducted under formal guidelines for company sponsored safety assessment of marketed medicines (SAMM). A non-randomised, non-interventional, observational design compared patients prescribed metered doses of salbutamol delivered by inhalers using either hydrofluoroalkane or chlorofluorocarbon as the propellant. Follow up was three months. SETTING: 646 general practices throughout the United Kingdom. SUBJECTS: 6614 patients with obstructive airways disease (1667 patient years of exposure). MAIN OUTCOME MEASURES: Proportions of patients who were: admitted to hospital for respiratory diseases, reported adverse side effects, or withdrew because of adverse affects. RESULTS: There were no significant differences between the hydrofluoroalkane (HFA 134a) and chlorofluorocarbon inhaler groups in relation to the proportions of patients admitted to hospital for respiratory diseases (odds ratio 0.75; 95% confidence interval 0.51 to 1.08) or the proportions who reported adverse events (1.01; 0.88 to 1.17). However, more patients using the hydrofluoroalkane inhaler than the chlorofluorocarbon inhaler withdrew because of adverse events (3.8% and 0.9% respectively). CONCLUSION: The hydrofluoroalkane inhaler was as safe as the chlorofluorocarbon inhaler when judged by hospital admissions and adverse affects. The study design successfully fulfilled the recommendations of the guidelines. Differences between postmarketing surveillance studies and randomised clinical trials in assessing safety were identified. These may lead to difficulties in the design of postmarketing surveillance studies.     

Relaxation of corpus cavernosum and raised intracavernous pressure by berberine in rabbit

1. In the present study, we have investigated the effect of berberine in rabbit isolated corpus cavernosum and measured the intracavernous pressure (ICP) change after intracavernosal injection of berberine in rabbit. 2. Berberine alone suppressed the basal tone and induced a concentration (0.1-100 microM)-dependent relaxation in phenylephrine (PE)-precontracted corpus cavernosum. 3. Tetrodotoxin (0.1 and 1 microM) treatment had no significant effect on the berberine-induced relaxation. Phentolamine (1 and 10 microM), propranolol (1 and 3 microM) and atropine (1 and 3 microM) were also without effect. These results suggest that berberine might cause relaxation of the cavernosal strip by direct action on the corpus cavernosum, not by a neuronal effect. Furthermore, muscarinic- and beta-adrenoceptors were not involved. 4. Berberine-induced relaxations were significantly reduced by endothelium removal and by exposure to L-NG-nitro arginine methyl ester (0.1 and 0.3 mM), but not indomethacin (30 microM). 5. In endothelium-deprived corpus cavernosal tissues, berberine-induced relaxations were significantly reduced in high K+ medium (KCl = 60 mM), by charybdotoxin (ChTX) and 4-aminopyridine (4-AP) but not by glibenclamide and apamin. 6. After intracavernous injection of berberine (1, 2, 3 and 5 mg kg(-1)), the ICP rose from 12.7+/-3.6 to 13.2+/-5.4, 25.3+/-6.1, 46.5+/-8.2, and 63.4+/-10.2 mmHg, respectively. The duration of tumescence ranged from 11.5 - 43.7 min. 7. The results show that berberine possesses a relaxant effect on rabbit corpus cavernosal tissues which is attributable to both endothelium-dependent and-independent properties. While the former component is apparently due to the release of NO from sinusoidal endothelium, the endothelium-independent mechanism involved in berberine relaxation is probably linked to ChTX- and 4-AP-sensitive K+ channel activation in the cavernosal vasculature.