Association of cyclophosphamide pharmacokinetics to polymorphic cytochrome P450 2C19.

Cyclophosphamide (CP), a widely used cytostatic, is metabolized by polymorphic drug metabolizing enzymes particularly cytochrome P450 (CYP) enzymes. Its side effects and clinical efficacy exhibit a broad interindividual variability, which might be due to differences in pharmacokinetics. CP-kinetics were determined in 60 patients using a global and a population pharmacokinetic model considering functionally relevant polymorphisms of CYP2B6, CYP2C9, CYP2C19, CYP3A5, and GSTA1. Moreover, metabolic ratios were calculated for selected CP metabolites, analyzed by (31)P-NMR-spectroscopy. Analysis of variance revealed that the CYP2C19*2 genotype influenced significantly pharmacokinetics of CP at doses

Sequence-dependent stability of intramolecular DNA triple helices.

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.

Structural heterogeneity in intramolecular DNA triple helices.

Oligodeoxynucleotides designed to form intramolecular triple helices are widely used as model systems in thermodynamic and structural studies. We now report results from UV, Raman and NMR experiments demonstrating that the strand polarity, which also determines the orientation of the connecting loops, has a considerable impact on the formation and stability of pyr x pur x pyr triple helices. There are two types of monomolecular triplexes that can be defined by the location of their purine tract at either the 5'- or 3'-end of the sequence. We have examined four pairs of oligonucleotides with the same base composition but with reversed polarity that can fold into intramolecular triple helices with seven base triplets and two T4 loops under appropriate conditions. UV spectroscopic monitoring of thermal denaturation indicates a consistently higher thermal stability for the 5'-sequences at pH 5.0 in the absence of Mg2+ ions. Raman spectra provide evidence for the formation of triple helices at pH 5 for oligomers with purine tracts located at either the 5'- or 3'-end of the sequence. However, NMR measurements reveal considerable differences in the secondary structures formed by the two types of oligonucleotides. Thus, at acidic pH significant structural heterogeneity is observed for the 3'-sequences. Employing selectively 15N-labeled oligomers, NMR experiments indicate a folding pattern for the competing structures that at least partially changes both Hoogsteen and Watson-Crick base-base interactions.

Influence of sequence-dependent cytosine protonation and methylation on DNA triplex stability.

To investigate cytosine protonation and its influence on the sequence-dependent thermal stability of DNA triplexes in detail, we have employed homo- and heteronuclear NMR experiments on specifically (15)N-labeled oligodeoxynucleotides that were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. These experiments reveal that cytosines in central positions of the triplex are significantly protonated even at neutral pH. However, semiprotonation points for individual cytosine bases as determined from pH-dependent measurements show considerable differences depending on their position. Thus, protonation is disfavored for adjacent cytosines or for cytosines at the triplex termini, resulting in a smaller contribution to the overall free energy of the triple helical system. In contrast, protonation of the base upon substitution of 5-methylcytosine for cytosine in the triplex third strand is only affected to a minor extent, and triplex stabilization by the methyl substituent is shown to primarily arise from stacking energies and/or hydrophobic effects.

Solution structure of the octamer motif in immunoglobulin genes via restrained molecular dynamics calculations.

The solution structure of the DNA decamer d(CATTTGCATC)-d(GATGCAAATG), comprising the octamer motif of immunoglobulin genes, is determined by restrained molecular dynamics (rMD) simulations. The restraint data set includes interproton distances and torsion angles for the deoxyribose sugar ring which were previously obtained by a complete relaxation matrix analysis of the two-dimensional nuclear Overhauser enhancement (2D NOE) intensities and by the quantitative simulation of cross-peaks in double-quantum-filtered correlated (2QF-COSY) spectra. The influence of torsion angles and the number of experimental distance restraints on the structural refinement has been systematically examined. Omitting part of the experimental NOE-derived distances results in reduced restraint violations and lower R factors but impairs structural convergence in the rMD refinement. Eight separate restrained molecular dynamics simulations were carried out for 20 ps each, starting from either energy-minimized A- or B-DNA. Mutual atomic root-mean-square (rms) differences among the refined structures are well below 1 A and comparable to the rms fluctuations of the atoms about their average position, indicating convergence to essentially identical structures. The average refined structure was subjected to an additional 100 ps of rMD simulations and analyzed in terms of average torsion angles and helical parameters. The B-type duplex exhibits clear sequence-dependent variations in its geometry with a narrow minor groove at the T3.A3 tract and a large positive roll at the subsequent TG.CA step. This is accompanied by a noticeable bend of the global helix axis into the major groove. There is also evidence of significant flexibility of the sugar-phosphate backbone with rapid interconversion among different conformers.

Deuterium NMR relaxation studies of peptide-lipid interactions.

A unique model membrane system composed of a synthetic amphiphilic peptide (Lys2-Gly-Leu16-Lys2-Ala-amide) and a specifically labeled phospholipid (1,2-[7,7-2H2]dipalmitoyl-sn-glycero-3-phosphocholine) has been studied by 2H NMR, using inversion recovery, quadrupolar echo, and modified Jeener-Broekaert sequences, from 213 to 333 K, at molar peptide concentrations of 0, 2, 4, and 6%. Analysis of the experiments, employing a density matrix treatment based on the stochastic Liouville equation, revealed information about the dynamic organization of the lipid in the model membrane system, whose phase behavior has been determined previously [Huschilt et al. (1985) Biochemistry 24, 1377-1386]. The dynamic organization is described in terms of segmental and molecular order parameters and in terms of correlation times corresponding to both internal and overall lipid motions. In the liquid crystalline phase, the molecular order parameter, SZZ, was observed to decrease slightly upon addition of peptide while the conformational order parameter corresponding to the seventh segment, SZ'Z', did not change for any concentration of peptide. In general, the gauche-trans isomerization rate in the middle of the chain was not observed to change upon peptide addition, whereas the whole body reorientational correlation times (tau R parallel and tau R perpendicular) increased by nearly an order of magnitude. The anisotropy ratio (tau R perpendicular/tau R parallel) decreased with peptide added. An additional motion which involves a jump about the axis of the sn-2 chain is also observed to be slowed down significantly in the presence of peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

The octamer motif in immunoglobulin genes: extraction of structural constraints from two-dimensional NMR studies.

Phase-sensitive two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlated (2QF-COSY) spectra were recorded at 500 MHz for the DNA duplex d(CATTTGCATC).d(GATGCAAATG), which contains the octamer element of immunoglobulin genes. Exchangeable and nonexchangeable proton resonances including those of the H5' and H5" protons were assigned. Overall, the decamer duplex adopts a B-type DNA conformation. Scalar coupling constants for the sugar protons were determined by quantitative simulations of 2QF-COSY cross-peaks. These couplings are consistent with a two-state dynamic equilibrium between a minor N- and a major S-type conformer for all residues. The pseudorotation phase angle P of the major conformer is in the range 117-135 degrees for nonterminal pyrimidine nucleotides and 153-162 degrees for nonterminal purine nucleotides. Except for the terminal residues, the minor conformer comprises less than 25% of the population. Distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities with the MARDIGRAS algorithm confirm the dependence of the sugar pucker on pyrimidine and purine bases. Averaging by fast local motions has at most small effects on the NOE-derived interproton distances.

Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior.

The influence of cholesterol on the dynamic organization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers was studied by deuteron nuclear magnetic resonance (2H NMR) using unoriented and macroscopically aligned samples. Analysis of the various temperature- and orientation-dependent experiments were performed using a comprehensive NMR model based on the stochastic Liouville equation. Computer simulations of the relaxation data obtained from phospholipids deuterated at the 6-, 13- and 14-position of the sn-2 chain and cholesterol labeled at the 3 alpha-position of the rigid steroid ring system allowed the unambiguous assignment of the various motional modes and types of molecular order present in the system. Above the phospholipid gel-to-liquid-crystalline phase transition, TM, 40 mol % cholesterol was found to significantly increase the orientational and conformational order of the phospholipid with substantially increased trans populations even at the terminal sn-2 acyl chain segments. Lowering the temperature continuously increases both inter- and intramolecular ordering, yet indicates less ordered chains than found for the pure phospholipid in its paracrystalline gel phase. Trans-gauche isomerization rates on all phospholipid alkyl chain segments are slowed down by incorporated cholesterol to values characteristic of gel-state lipid. However, intermolecular dynamics remain fast on the NMR time scale up to 30 K below TM, with rotational correlation times tau R parallel for DMPC ranging from 10 to 100 ns and an activation energy of ER = 35 kJ/mol. Below 273 K a continuous noncooperative condensation of both phospholipid and cholesterol is observed in the mixed membranes, and at about 253 K only a motionally restricted component is left, exhibiting slow fluctuations with correlation times of tau R perpendicular greater than 1 microsecond. In the high-temperature region (T greater than TM), order director fluctuations are found to constitute the dominant transverse relaxation process. Analysis of these collective lipid motions provides the viscoelastic parameters of the membranes. The results (T = 318 K) show that cholesterol significantly reduces the density of the cooperative motions by increasing the average elastic constant of the membrane from K = 1 x 10(-11) N for the pure phospholipid bilayers to K = 3.5 x 10(-11) N for the mixed system.

H-NMR studies on d(GCTTAAGC)2 and its complex with berenil.

Two-dimensional (2D) 1H-NMR spectroscopy has been used to analyze the structure of d(GCTTAAGC)2 and its interaction with berenil in solution. Nuclear Overhauser enhancement connectivities enabled sequential assignments of nearly all proton resonances in the self-complementary octamer duplex and demonstrated that the oligonucleotide is primarily in a B-type conformation. No major conformational changes were observed by the addition of berenil, but proton resonances of the two adenosine nucleotides shifted substantially. Intermolecular nuclear Overhauser effects between berenil and the DNA duplex revealed that the drug binds via the minor groove of d(GCTTAAGC)2 in the A.T-base-pair region. At 18 degrees C the twofold symmetry of the duplex is preserved on berenil binding. However, strongly shifted proton resonances broadened significantly. A model is proposed for the berenil-d(GCTTAAGC)2 complex involving fast exchange of berenil between two equivalent symmetry-related binding sites, which span the 5'-TAA-3' region and are asymmetrically disposed with respect to the dyad axis of the duplex. These results are compared with previous studies on the berenil-d(GCAATTGC)2 complex.

Pathogen-related oral spirochetes from dental plaque are invasive.

Spirochetes that share pathogen-restricted antigens with Treponema pallidum subsp. pallidum have been identified in dental plaque and diseased gingival tissues, but it is not known whether these spirochetes possess virulence characteristics. In this study, plaque spirochetes were able to transmigrate a tissue barrier in vitro and were identified on the other side by using monoclonal antibodies specific for pathogen-restricted determinants from T. pallidum subsp. pallidum. This invasive capability is shared with T. pallidum subsp. pallidum, but cultured oral and intestinal treponemes did not perforate the tissue barrier. Cocultures indicated that invasive treponemes do not create opportunities for cultivable oral treponemes to cross the barrier. These findings indicate that gingival tissues may be a port of entry for previously unrecognized invasive spirochetes in humans.

Pathogen-related spirochetes identified within gingival tissue from patients with acute necrotizing ulcerative gingivitis.

The purpose of this investigation was to determine whether monoclonal antibodies against pathogen-restricted antigens of Treponema pallidum subsp. pallidum could be used as probes for spirochetes in diseased gingival tissue from subjects with acute necrotizing ulcerative gingivitis. A biotin-streptavidin system was used to identify spirochetes bound by monoclonal antibodies in cryostat sections of tissue. Twelve of 16 tissue samples from diseased sites, but none of 8 tissue specimens from healthy sites, reacted with pathogen-restricted antibodies. Organisms were found in intact epithelium and connective tissues adjacent to ulcers. Staining intensity was often high in perivascular locations and around vesicular spaces. Monoclonal antibodies to Bacteroides gingivalis and Treponema denticola were each reactive with diseased gingival tissues, but staining was usually restricted to ulcerated areas. These studies extend recent observations that showed that subjects with acute necrotizing ulcerative gingivitis had both pathogen-related spirochetes in dental plaque and serum immunoglobulin G to pathogen-restricted antigens on T. pallidum subspecies, suggesting that pathogen-related spirochetes may be associated with the pathogenesis of certain periodontal diseases.

Identification of spirochetes related to Treponema pallidum in necrotizing ulcerative gingivitis and chronic periodontitis.

BACKGROUND: Spirochetes are commonly associated with periodontal disease, but it is not known whether these treponemes are pathogenic or merely opportunistic. We sought to determine whether spirochetes present in periodontal disease share antigens thought to be unique to spirochetes that are known pathogens. METHODS: We examined dental plaque from 24 healthy subjects, from ulcerative sites in 17 patients with ulcerative gingivitis, and from areas of involvement in 19 patients with chronic periodontitis, using an immunocyto-chemical technique with monoclonal antibodies against pathogen-specific determinants on 47-kd and 37-kd molecules from Treponema pallidum subspecies pallidum. Serum was tested against T. pallidum by immunoblotting and by serologic assays for syphilis. RESULTS: Spirochetes with a pathogen-specific epitope on a 47-kd molecule were not found in plaque samples from any of the 24 healthy subjects, but they were identified in plaque samples from 11 of 17 patients with ulcerative gingivitis (P less than 0.001) and from 10 of 19 patients with periodontitis (P less than 0.01). Monoclonal antibodies directed against a 37-kd molecule reacted with spirochetes in plaque samples from 1 of 14 controls, from all 11 patients with gingivitis from whom samples could be obtained (P less than 0.001), and from 14 of 19 patients with periodontitis (P less than 0.001). Five of 18 normal subjects had IgG against 47-kd and 37-kd molecules, but none had IgG against 14-kd or 12-kd molecules from T. pallidum subspecies pallidum. Among 19 patients with ulcerative gingivitis, IgG was identified against 47-kd molecules in 15, against 37-kd molecules in 12, against 14-kd molecules in 4, and against 12-kd molecules in 15. CONCLUSIONS: The spirochetes found in dental plaque from patients with ulcerative gingivitis or chronic periodontitis have antigens that are thought to be unique to pathogenic treponemes. This close antigenic relation suggests that T. pallidum or a closely related organism may be involved in the pathogenesis of periodontal disease.

Self-restraint: a review of etiology and applications in mentally retarded adults with self-injury.

Self-restraint has been noted in studies of self-injurious behavior (SIB) in mentally retarded individuals. These studies have investigated self-restraint in the context of SIB, and as a result it has been difficult to isolate the function of self-restraint within this complex clinical situation. This article summarizes and critically analyzes the literature on self-restraint, particularly its identification, etiology, modification, and relationship to SIB. At present there is some empirical evidence to suggest that self-restraint is negatively reinforced by the termination or avoidance of SIB. Other studies have demonstrated that self-restraint can develop stimulus control properties for the absence of SIB. Finally, it has been suggested that self-restraint may function independently of SIB and be maintained by escape from demands or by conditioned reinforcement. Clinical concerns include the need for a functional analysis of self-restraint, the undesirable effects on SIB of reducing self-restraint, and the necessity of replacing self-restraint topographies with ones which do not interfere with daily activities.

Human brain 6-phosphogluconate dehydrogenase: purification and kinetic properties.

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.