Global leukocyte DNA methylation is not altered in euthymic bipolar patients.

BACKGROUND: Bipolar disorder is a complex disorder hypothesized to involve an interaction of multiple susceptibility genes and environmental factors. The environmental factors may be mediated via epigenetic mechanisms such as DNA methylation. Since a different extent of DNA methylation has recently been reported in lymphoblastoid cells derived from monozygotic twins discordant for bipolar disorder, we hypothesized that bipolar patients exhibit a different extent of leukocyte global DNA methylation compared with healthy controls. METHODS: DNA was extracted from peripheral blood leukocytes of 49 euthymic bipolar patients and 27 matched healthy controls. Percent of global genome DNA methylation was measured using the cytosine-extension method. Plasma homocysteine levels were measured by HPLC. RESULTS: Leukocyte global DNA methylation did not differ between bipolar patients [62.3%+/-18.0 (S.D)] and control subjects (63.9%+/-14.6), p=0.70. Bipolar patients' plasma homocysteine levels (11.5 microM+/-4.8) did not differ from those of healthy controls (11.4+/-2.9), p=0.92. LIMITATIONS: The assay we used, based on restriction by methylation-sensitive/insensitive enzymes followed by a radioactive DNA polymerase reaction was approved to accurately measure global DNA methylation, but has technical limitations i.e. restriction enzymes do not cleave all potential methylation sites in the genome and restriction sites may be altered by mutations or polymorphisms. CONCLUSIONS: The lack of difference in leukocyte global DNA methylation between euthymic bipolar patients and healthy controls does not rule out the possibility that altered methylation of specific promoter regions is involved in the etiology of the disorder.

No association between global leukocyte DNA methylation and homocysteine levels in schizophrenia patients.

Meta-analysis recently suggested that a 5 muM increase in homocysteine is associated with a 70% higher risk for schizophrenia. Elevated homocysteine is reported to alter macromolecule methylation. We studied whether elevated plasma homocysteine levels in schizophrenia are associated with altered leukocyte global DNA methylation. DNA was extracted from peripheral blood leukocytes of 28 schizophrenia patients vs. 26 matched healthy controls. Percent of global genome DNA methylation was measured using the cytosine-extension method. Homocysteine levels were higher in schizophrenia patients than in controls. No difference in global DNA methylation between schizophrenia patients and control subjects was found (74.0%+/-14.8 vs. 69.4+/-22.0, p=0.31). A significant interaction between diagnosis and smoking on DNA methylation was obtained (F=6.8, df=1,47, p=0.032). Although leukocytes may be a useful cell model to evaluate epigenetic changes such as global DNA methylation in brain, future studies should compare global DNA methylation in peripheral tissue vs. brain in laboratory animals.

Detection of frequency resonance energy transfer pair on double-labeled microsphere and Bacillus anthracis spores by flow cytometry.

Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare. Fluorescence methods combined with immunodiagnostic methods are the most common. To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method. FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET. Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method. This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system. As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores. The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B. anthracis exosoporium components. The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor. On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (E(q) = 35%) and appearance of the alexa594 fluorescence (E(s) = 22%), as detected by flow cytometry analysis. The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B. thuringiensis subsp. israelensis and B. subtilis. This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B. anthracis spores by a factor of 10 with respect to B. thuringiensis subsp. israelensis and a factor of 100 with respect to B. subtilis as control spores.

Light-dependent oxygen consumption in bacteriochlorophyll-serine-treated melanoma tumors: on-line determination using a tissue-inserted oxygen microsensor.

Successful application of anticancer therapy, and especially photodynamic therapy (PDT) mediated by type II (PDTII) processes, depends on the oxygen content within the tumor before, during and after treatment. The high consumption of oxygen during type II PDT imposes constraints on therapy strategies. Although rates of oxygen consumption and repletion during PDTII were suggested by theoretical studies, direct measurements have not been reported. Application of a novel oxygen sensor allowed continuous and direct in situ measurements (up to a depth of 8-9 mm from the tumor surface and for several hours) of temporal variations in the oxygen partial pressure (pO2) during PDT. Highly pigmented M2R mouse melanoma tumors implanted in CD1 nude mice were treated with bacteriochlorophyll-serine (Bchl-Ser; a new photodynamic reagent) and were subjected to fractionated illumination (700 < lambda < 900 nm) at a fluence rate of 12 mW cm-2. This illumination led to total oxygen depletion with an average consumption rate of 7.2 microM(O2) s-1. Spontaneous reoxygenation (at an average rate of 2.5 microM(O2)/s) was observed during the following dark period. These rates are in good agreement with theoretical considerations (Foster et al., Radiat. Res. 126, 296, 1991 and Henning et al., Radiat. Res. 142, 221, 1995). The observed patterns of oxygen consumption and recovery during prolonged periods of light/dark cycles were interpreted in terms of vasculature damage and sensitizer clearance. The presented data support the previously suggested advantages of fractionated illumination for type II photodynamic processes.

Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite.

Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.

Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core.

Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.

The third epidemic--multidrug-resistant tuberculosis.

We recently observed a striking increase in multidrug-resistant tuberculosis (MDR-TB) among patients admitted to the Chest Service at Bellevue Hospital Center in New York. We reviewed the laboratory susceptibility test results of 4,681 tuberculosis (TB) cases over the past 20 years, Combined resistance to isoniazid and rifampin increased from 2.5 percent in 1971 to 16 percent in 1991 with higher rates noted for individual drugs. We reviewed the medical records of 100 patients with drug-resistant TB, finding that these individuals were predominantly less than 40 years of age, minority, male, jobless, undomiciled, with a high percentage of drug abuse and human immunodeficiency virus infection. We conclude that the epidemics of AIDS and TB are complicated by a third epidemic of MDR-TB. This third epidemic requires urgent attention to achieve more rapid diagnosis, to develop new therapeutic regimens, and to address the social and hospital environment ot care for these individuals.

Mechanism of inhibition of hemoglobin S polymerization by cyanate.

We have studied the effect of carbamylation on hemoglobin S (HbS) polymerization with the use of a new quantitative gelling technique. HbS gels fromed in the presence of nitrogen and dithionite (with or without carbon monoxide) at pH 7 in 0.15M phosphate were separated into sol and gel phases by centrifugation at 130,000 g. Hb concentration to the sol phase ([Hb]sol) of nonligated HbS was found to be constant (10.5 +/- 092 mM heme) over a range of original Hb concentrations from 11 to 17 mM at 24degrees C. This suggests that the HbS sol-gel equilibrium behaves as simple two-phase system. Increasing levels of total carbamylation from 0.65 to 3.65 moles CNO-/mole Hb tetramer (0.36 to 2.2 moles N-terminal/mole Hb4) progressively increases [Hb]sol. Specific activity of 14CNO-HbS was similar in the sol and gel phases, whereas COHbS appeared to be completely excluded from polymer structure of the gel. A comparison of the solubility of uncarbamylated and heavily carbamylated HbS at Co saturations ranging from 3 to 61 percent showed that the larges difference in [Hb]sol occured at the lowest ligand saturation rather than at intermediate states of ligation. Inhibition of HbS polymerization by carbamylation under these conditions, therefore, is not primarily the result of an effect on the T in equilibrium R comformational equilibrium. Our findings indicate that cyanate can interfere with HbS polymerization directly, possibly by alteration of surface binding sit(s) which are critical to aggregation. This direct action of cyanate may contribute significantly to the hematologic improvement achieved by cyanate treatment in sickle cell disease.