Metabolic activation of sulfur mustard leads to oxygen free radical formation.

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 µM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.

Therapeutic effects of gelatin matrix-embedded IL-12 gene-modified macrophages in a mouse model of residual prostate cancer.

We evaluated the potential use of intraoperative gelatin matrix hemostatic sealant (GMHS; FloSeal; Baxter Healthcare) embedded with macrophages (Mphi) transduced with murine interleukin (IL)-12 recombinant adenoviral vector (G/Mphi/AdmIL-12) for prevention of recurrence of prostate cancer following radical prostatectomy. Application of G/Mphi/AdmIL-12 resulted in significant suppression of tumor growth and spontaneous lung metastases, a statistically significant survival advantage of the G/Mphi/AdmIL-12-treated animals, more efficient trafficking of Mphi to lymph nodes draining from the prostate and generation of systemic natural killer cell activity and tumor-specific cytolytic T lymphocyte responses compared to the controls in a preclinical mouse model of residual prostate cancer. Our data recommend this treatment as a novel adjuvant for prevention of local recurrence of prostate cancer following radical prostatectomy.

TNF-alpha expression patterns as potential molecular biomarker for human skin cells exposed to vesicant chemical warfare agents: sulfur mustard (HD) and Lewisite (L).

Studies were conducted to examine the effect of two vesicant chemical warfare agents (VCWA), one of them an arsenical, on cytokine gene expression in normal human epidermal keratinocyte (NHEK) cells. We tested 2,2'-dichlorethylsulfide (sulfur mustard, military designation HD) and 2,chlorovinyldichloroarsine (Lewisite, military designation L), which have significant differences in their chemical, physical, and toxicological properties. Human tumor necrosis factor-alpha (hTNF-alpha) cytokine was detected by using the enzyme-linked immunosorbent assay, a protein multiplex immunoassay, Luminex100, and reverse transcription-polymerase chain reaction (RT-PCR). The messenger RNA expression of hTNF-alpha was determined to provide a semi-quantitative analysis. HD-stimulated NHEK induced secretion of hTNF-alpha in a dose-dependent manner. Dose response effect of Lewisite decreased hTNF-alpha levels. Time-response data indicated that the maximum response for HD occurred at 24 h with an associated cytotoxic concentration of 10(-4) mol/L. NHEK cells stimulated with 10(-4) mol/L HD for 24 h at 37 degrees C increased detectable levels of hTNF-alpha from 5 to 28 ng/ml at an index of cell viability between 85 to 93% as detected by Luminex100. Our results indicated that the increased levels of hTNF-alpha by HD are dependent on the primary cultures, cell densities, and chemical properties of the stimulation. Lewisite under the same conditions as HD caused a reduction of hTNF-alpha from control levels of 1.5 ng/ml to 0.3 ng/ml after stimulation (10(-4) mol/L), with an index of cell viability of reverse similar 34%. We analyzed the transcriptional of hTNF-alpha gene and found that HD (10(-6) to 10(-4) mol/L) activates hTNF-alpha gene in cultured NHEK and that L at 10(-6) to 10(-4) mol/L markedly reduces hTNF-alpha gene. We conclude that the pro-inflammatory mediator, hTNF-alpha, could be a potential biomarker for differentiating between exposure of HD or L.

Solution structure of a nucleic acid photoproduct of deoxyfluorouridylyl-(3'-5')-thymidine monophosphate (d-FpT) determined by NMR and restrained molecular dynamics: structural comparison of two sequence isomer photoadducts (d-U5p5T and d-T5p5U).

Acetone-sensitized irradiation using UV-B (sun lamp, lambda max = 313 nm) of deoxyfluorouridylyl-(3'-5')-thymidine monophosphate (d-FpT, F = fluorouracil), produces two major photoproducts, the cis-syn cyclobutane-type photodimer and a defluorinated (5-5) photoadduct, d-U5p5T. Product distribution is dependent on the pH of the irradiation solution, as was the case of irradiated d-TpF. At high pH (8-10) the (5-5) photoadduct is the major photoproduct. Irradiation of d-FpT shows a much faster photodegradation rate than the sequence isomer d-TpF. Multinuclear NMR experiments establish the formation of (5-5) covalent bonding between the C5 (d-U5p-, where the fluorine had been) and the C5 (-p5T) and the C6 (-p5T) acquires an OH group. NOE interproton distances and dihedral angles derived from J coupling analysis are constrained to refine model structures of d-U5p5T in restrained molecular dynamics calculations. The resultant structures obtained show 5S-6S as the most chiralities of the C5 and C6 atoms of the thymine, which is the opposite chirality to the corresponding atoms in the sequence isomer d-T5p5U. The orientation of the C5 substituents (-p5T fragment), the CH3 and the uracil are pseudo-axial and pseudo-equatorial respectively. Glycosidic angles are in the anti regions for both the d-U5p- and -p5T residues. Averaged backbone conformations of the two photoadducts, d-U5p5T and d-T5p5U, are similar, although the overall structure of d-U5p5T appears much more flexible than that of d-T5p5U. In particular, the sugar conformations of the 5'-end residues show a remarkable difference in flexibility.

Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins.

The conformational preference of the candidacidal C-terminal 16 residue fragment (9-24; G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) of salivary histatin 5 was examined in water, methanol, and dimethyl sulfoxide solutions using 500 MHz two-dimensional-NMR. Fourier transform infrared and CD spectroscopy were used to delineate its membrane-bound conformation in lipid vesicles. The peptide backbone and side-chain proton resonance assignments were accomplished by two-dimensional total correlated and nuclear Overhauser effect (NOE) spectra. The coupling constant (JNH-C alpha H) values determined from the double quantum-filtered correlated spectra, temperature coefficients of NH chemical shifts (d delta/dT), 1H/2H exchange rates on amide resonances, and the set of NOE connectivities were used to delineate backbone conformational features. The high JNH-C alpha H values (> or = 7.4 Hz), absence of any characteristic NH-NH (i, i + 1) or C alpha H-C beta H (i, i + 3) NOE connectivities, high d delta/dT values (> or = 0.004), and the fast 1H/2H amide exchange suggest that the histatin peptide favors unfolded random conformations in aqueous solution at pH 3.8. In contrast, the JNH-C alpha H values (< or = 6.5 Hz), slow 1H/2H exchange, low d delta/dT values (< or = 0.003) observed for amide resonances of residues 5-16, and the characteristic NH-NH (i, i + 1), C alpha H-C beta H (i, i + 3) NOE connectivities, provide evidence for the presence of largely alpha-helical conformations in dimethyl sulfoxide, which mimics the polar aprotic membrane environment. In methanolic solutions, 3(10)-helical conformations could exist as a minor population together with the major alpha-helical conformations. Fourier transform infrared spectroscopy and CD data indicate that lipid environments such as dimyristoylphosphatidylcholine vesicles could induce the peptide to fold into predominantly alpha-helical conformation. The results suggest that in dimethyl sulfoxide and dimyristoylphosphatidylcholine vesicles the candidacidal domain of salivary histatin 5 prefers a largely helical conformation, which could facilitate its interaction with the membrane of Candida albicans. The mechanism of antimicrobial action of this class of polypeptides appears to involve primarily electrostatic and hydrogen-bonding interaction of cationic and polar residues with the head groups of the plasma membranes of target cells.

Serum iron abnormalities in neuroleptic-induced akathisia in schizophrenic patients.

Twenty-two schizophrenic patients (DSM-III-R criteria) with clinically significant akathisia were matched with 22 schizophrenic patients without akathisia on the following variables: age, sex, diagnosis, duration of illness, and current treatment. Both groups were assessed using a variety of clinical rating scales and several parameters of serum iron status. The akathisic patients showed greater severity of clinical psychopathology, particularly positive symptoms, and an excess of extrapyramidal side-effects. We were unable to confirm any association between low serum iron and neuroleptic-induced akathisia in our sample of community-based patients.

Solution structure of nucleic acid photoadduct, deoxy-m5HO6-uridylyl(5-5)(3'-5')deoxyuridine by NMR and restrained molecular dynamics.

Sensitized UV-B irradiation (sunlamps) of the dinucleoside monophosphate, d-TpF (F = fluorouracil), produces the usual cyclobutane-type photodimer and an additional defluorinated 5-5 photoadduct, d-T5p5U. In d-T5p5U, the original C5 = C6 structure is modified such that the C5 (d-T5p-) is covalently bonded with the C5 (-p5U) (where the fluorine had been) and the C6 (d-T5p-) acquires an OH group. 2D NOE data and the results of J-coupling analysis are used as constraints to refine structures of d-T5p5U in restrained molecular dynamics calculations. The structures obtained show the most probable chiralities of the C5 and C6 atoms of the Thy-portion to be 5R and 6R, respectively. The orientation of the CH3- and uracil-groups are pseudo-axial and pseudo-equatorial, respectively, with respect to the C5 atom. Glycosidic angles are high-anti and anti for the d-T5p- and the -p5U residue, respectively. C3'-endo like sugar puckering is predominant in the d-T5p- residue while C2'-endo like puckering is predominant at the -p5U residue.

UV irradiation of nucleic acids: characterization of photoproducts of thymidylyl-(3'-->5')-2'-deoxy-5-fluorouridine.

The acetone-sensitized irradiation using UV-B (ultraviolet light, 280-320 nm; sunlamps) of thymidylyl(3'-->5')deoxyfluorouridine monophosphate produces two main photoproducts. The distribution of these photoproducts is dependent on the pH of the irradiation solution. At pH 6, the cis-syn cyclobutane-type photodimer is the major product, whereas at high pH (8-10) a photoadduct is the major product. These photoproducts have been identified and structurally characterized by H-1 and C-13 NMR spectroscopy. The photoadduct arises from defluorination of the 5-fluorouracil moiety. The structure of the photoadduct maintains the sugar-phosphate backbone of the starting material (d-TpF), and contains a saturated thymine moiety with an added Thy(C6-hydroxyl) and a Thy(C5)-(C5)Ura covalent bond.

Rationalizing neuroleptic polypharmacy in chronic schizophrenics: effects of changing to a single depot preparation.

This study investigated the effects of transferring patients on combined depot and oral neuroleptics to a single depot preparation; a secondary objective was to assess the effects of transferring patients from one depot neuroleptic to another. It was found that, whereas transferring from one depot preparation (flupenthixol) to another (fluphenazine) had no clear disadvantage for the patients, changing over from a combined oral and depot (fluphenazine) regimen to equivalent doses of depot alone resulted in an unacceptably high rate of relapse. The reasons for this may relate to either the unique pharmacokinetics of these drugs or subtle qualitative differences between them. It is suggested that caution is necessary whenever attempts are made to rationalize polypharmacy in schizophrenic patients.

Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva.

The major components of human submandibular-sublingual saliva (HSMSL) are mucins, amylases, cystatins, proline-rich proteins and statherin. Structure-function studies of these molecules have been hampered by the small amounts of purified materials that can be isolated from human secretions. The present study describes an integrated purification protocol for the large-scale preparation of many of these molecules. To dissociate partially heterotypic complexes among salivary molecules, HSMSL was initially fractionated into four pools by gel filtration with 6 M-guanidine hydrochloride. Subsequent fractionation of these four pools by gel-filtration and ion-exchange chromatography resulted in the purification of high- and low-Mr mucins, neutral and acidic cystatins, acidic and basic proline-rich proteins and statherin. Many variants or isoforms of these salivary molecules have been identified and biochemically characterized. Biochemical studies indicated that the low-Mr mucin exists as two isoforms which vary in their sialic acid to fucose ratios. Three isoforms of acidic cystatin S were characterized which differ in their phosphate content. Two isoforms of a basic proline-rich peptide were identified; the smaller peptide was a truncated form missing the first seven amino acids.

Crystal structure and solution conformation of S,S'-bis(Boc-Cys-Ala-OMe): intramolecular antiparallel beta-sheet conformation of an acyclic cystine peptide.

The conformation of the acyclic biscystine peptide S,S'-bis(Boc-Cys-Ala-OMe) has been studied in the solid state by x-ray diffraction, and in solution by 1H- and 13C-nmr, ir, and CD methods. The peptide molecule has a twofold rotation symmetry and adopts an intramolecular antiparallel beta-sheet structure in the solid state. The two antiparallel extended strands are stabilized by two hydrogen bonds between the Boc CO and Ala NH groups [N...O 2.964 (3) A, O...HN 2.11 (3) A, and NH...O angle 162 (3) degrees]. The disulfide bridge has a right-handed conformation with the torsion angle C beta SSC beta = 95.8 (2) degrees. In solution the presence of a twofold rotation symmetry in the molecule is evident from the 1H- and 13C-nmr spectra. 1H-nmr studies, using solvent and temperature dependencies of NH chemical shifts, paramagnetic radical induced line broadening, and rate of deuterium-hydrogen exchange effects on NH resonances, suggest that Ala NH is solvent shielded and intramolecularly hydrogen bonded in CDCl3 and in (CD3)2SO. Nuclear Overhauser effects observed between Cys C alpha H and Ala NH protons and ir studies provide evidence of the occurrence of antiparallel beta-sheet structure in these solvents. The CD spectra of the peptide in organic solvents are characteristic of those observed for cystine peptides that have been shown to adopt antiparallel beta-sheet structures.

Radio receptor assay of serum neuroleptic levels in psychiatric patients.

The radioreceptor assay for the measurement of neuroleptic drugs in serum has been used to study the relationship between dose and serum level in schizophrenic patients receiving these drugs. The assay was found to be reproducible and capable of detecting neuroleptics in the sera of patients receiving a range of both oral and depot drugs, with the exception of trifluoperazine spansules. Linear correlations were obtained between daily dose and serum level for each drug both in individual patients on different doses and between patients on a stable dose. Extrapyramidal side effects were related to the serum neuroleptic level within, but not between, patients. The assay may be of use in clinical practice, including the assessment of compliance or poor response to neuroleptics.