Presence Of Deuterium Oxide Research Articles

Phototoxicity of Lumidoxycycline

Doxycycline (DOTC) is a photosensitizing drug whose mechanism of phototoxicity is complicated by the large variety of stable photoproducts formed. To assess the role of a DOTC photoproduct, lumidoxycycline (LuDOTC), in the photosensitization mechanism of DOTC, MGH-U1 human bladder carcinoma cells were treated in vitro with either DOTC or LuDOTC, and irradiated with the 351-nm emission of an argon-ion laser. Both DOTC and LuDOTC were phototoxic and caused radiant-exposure-dependent inhibition of cellular incorporation of tritiated thymidine. On an absorbed-photon basis, DOTC was about five times as phototoxic as LuDOTC. Cellular uptake of DOTC was about five times as great as that of LuDOTC. Epifluorescence microscopy showed localization of LuDOTC predominantly within cellular membranes, particularly of mitochondria, as well as a low level of LuDOTC fluorescence diffusely within the cytoplasm. Epifluorescence microscopy of cells labeled with the mitochondrial probe, rhodamine 123, showed mitochondrial fragmentation and altered mitochondrial membrane integrity after LuDOTC photosensitization; these effects depended on radiant exposure and were partially reversible by 24 h after irradiation. For both DOTC and LuDOTC, phototoxicity was increased by irradiation in the presence of deuterium oxide and decreased in the presence of sodium azide, effects consistent with an important mechanistic role for singlet oxygen, O2(1 delta g), in the injury. In solution, LuDOTC and DOTC had similar quantum yields for generation of O2(1 delta g) as measured by time-resolved spectroscopy and by O2(1 delta g) trapping. LuDOTC was photostable in solution, but DOTC underwent significant photodegradation. These data demonstrate that DOTC photo-products such as LuDOTC have significant photobiologic activity and may play an important role in the phototoxicity mechanism of DOTC.

Investigation of induced circular dichroism of benzo(a)pyrene cyclodextrin complexes

This study focuses on the inclusion complexes of α-,β- andγ-cyclodextrins with benzo (a) pyrene, as indicated by induced circular dichroism data. The benzo(a)pyrene complex exhibits a significant induced ellipticity in the presence ofγ-cyclodextrin, while the other two cyclodextrins did not produce a significant induced circular dichroism signal. In the presence of deuterium oxide, which is larger than the water molecule, the ellipticity is larger. The changes in ellipticity with increasing cyclodextrin concentration are observed to follow the changes in equilibrium of the pyrene-γ-cyclodextrin system. The study of these systems using circular dichroism measurements yields a method of estimating the possible stoichiometry of this complex.

A novel .alpha.-proton exchange reaction catalyzed by Escherichia coli methionyl-tRNA synthetase

The Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) was shown to catalyze alpha-carbon hydrogen-deuterium exchange of L-selenomethionine, L-methionine, L-ethionine, and L-norleucine in the presence of deuterium oxide. The rate of alpha-proton exchange for L-methionine was shown to be linear with respect to delta MTS concentration. The exchange reaction showed saturation kinetics with apparent Km values of 21 and 4 mM in the absence and presence of saturating adenosine concentrations, respectively. As expected, delta MTS did not catalyze alpha-proton exchange of D-methionine since the enzyme has been shown to be specific for L-amino acids. In the absence of enzyme or in the presence of an equivalent concentration of Zn2+, no hydrogen-deuterium exchange was detected. The exchange reaction was not observed with L-methioninol, an analogue of L-methionine lacking the carboxylate group. These results suggest that the alpha-carboxylate group is a requirement for the delta MTS-catalyzed exchange reaction. The E. coli methionyl-tRNA synthetase (MTS) has previously been shown to be a zinc metalloprotein [Posorske, L. H., Cohn, M., Yanagisawa, N., & Auld, D. S. (1979) Biochim. Biophys. Acta 576, 128]. On the basis of the structural and mechanistic information available on MTS, we propose that the enzyme-bound zinc coordinates the carboxylate of the amino acid, while a base on the enzyme is responsible for exchange of the alpha-proton. The role of the enzyme-bound metal is to render the alpha-proton more acidic through coordination of the carboxylate group.(ABSTRACT TRUNCATED AT 250 WORDS)

Total synthesis of analogues of the β-lactam antibiotics. Part 5. 3-Thiacepham-4-exo-carboxylates and their 1,1,3,3-tetraoxides

t-Butyl hydroxy{4-[(Z)-2-(methoxycarbonyl)vinylthio]-2-oxoazetidin-1-yl}acetate (19a) was transformed into t-butyl 2-endo-methoxycarbonylmethyl-3-thiacepham-4-exo-carboxylate (20a) by sequential reactions involving thionyl chloride, potassium thioacetate, and cyclohexylamine. Oxidation of compound (20a) with potassium permanganate gave the 1,1,3,3-tetraoxide (23a), the structure of which was established by X-ray crystallography. The exceptional acidity of the 2- and 4-hydrogen atoms of compound (23a) was revealed by their replacement with deuterium atoms in the presence of deuterium oxide and with methyl groups in the presence of diazomethane. Cleavage of the t-butyl ester function of the thiacepham tetraoxide (23a) was effected by trifluoroacetic acid but the resultant acid (23b) underwent a rapid decarboxylation in water to give 2-endo-methoxycarbonylmethyl-3-thiacepham 1,1,3,3-tetraoxide (26a).

Identification of human lung mast cell kininogenase as tryptase and relevance of tryptase kininogenase activity

We have described previously the IgE-mediated release of kininogenase activity from purified human lung mast cells. Using supernatant fractions from mast cells stimulated with anti-IgE in the presence of deuterium oxide, we have purified this kininogenase to homogeneity by gel filtration and heparin-agarose chromatography and have demonstrated that it is identical to tryptase, the major neutral protease of human lung mast cells. Thus, tryptase and kininogenase activities co-chromatographed through both purification steps with equivalent yields. The final purified kininogenase was free of detectable chymotryptic and carboxypeptidase activities and was identified as tryptase on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition and inhibition profile. Three such preparations of tryptase were all capable of releasing kinin from each of two different preparations of purified, single-chain, human low molecular weight kininogen. Interestingly, kinin generation was optimal at pH 5.5 and was enhanced by heparin, which has been reported to stabilize tryptase. SDS-PAGE analysis of kininogen hydrolysis by tryptase revealed the formation of a diffusely stained region in the molecular weight range of 60,000–65,000, rather than a discrete heavy chain band. Under optimal conditions, the three tryptase preparations released 10–12 μg kinin/hr/mg but released only 2 μg kinin/hr/mg at pH7.2. HPLC analysis revealed that the kinin released was bradykinin. We conclude that the kininogenase activity from human lung mast cells is attributable to tryptase. The unique pH optimum of this reaction of a serine protease, however, raises doubts as to the physiologic significance of this activity.

Mitochondrial Phototoxicity Sensitized by Doxycycline in Cultured Human Carcinoma Cells

Cultured MGH-U1 (human urinary bladder carcinoma) cells were treated with doxycycline (DOTC) and long-wave UV radiation (UVA). At UVA doses of 1 J/cm2 and above, the cells showed mitochondrial damage, reflected by altered localization of the fluorescent probe rhodamine-123, and striking vacuolization of the cytoplasm. Cell membrane integrity, as monitored by exclusion of ethidium bromide, was maintained for several hours after mitochondrial damage was evident. These changes were potentiated by irradiation in the presence of deuterium oxide, and diminished by irradiation in the presence of sodium azide. Addition of catalase, superoxide dismutase, or mannitol did not alter the damage threshold. These data indicate that the mitochondrion is an earlier target of DOTC photosensitization than the cell membrane. The critical photochemistry appears to involve singlet oxygen.

Polyfluorocycloalkenes. Part XIX. Some reactions and compounds from 1,2-bis(trifluoromethyl)octafluorocyclohexene

The title compound (A) gave a dichloro-adduct (D), which with lithium aluminum hydride yielded a complex mixture, from which a trace of trans 1H, 2H-1, 2-bis(trifluoromethyl)octafluorocyclohexane (E) was isolated. Catalytic hydrogenation of olefin (A) afforded cis 1H,2H-1,2-bis(trifluoromethyl)octafluorocyclohexane (C), and a small amount of 6H-1,6-bis(trifluoromethyl)heptafluorocyclohexene (B). Dehydrofluorination of the cis dihydride (C) by aqueous potash gave olefin (B) and 2,3-bis(trifluoromethyl)hexafluorocyclohexa-1,3-diene (G): fluorination of (G) by cobalt(III) fluoride gave back (A). Fluorination of compounds (C) and (B) afforded cis and trans 1H-1,2-bis(trifluoromethyl)nonafluorocyclohexane (J and H respectively). Both (H) and (J) were dehydrofluorinated to give olefin (A) exclusively, i.e. fluorine was lost preferentially from the tertiary →CF group. Ammonia and (A) gave 1,3-diamino-2-cyano-3-trifluoromethylhexafluorocyctohexene (K). Some hydro-polyfluoro-cyclohexanes took up small proportions of deuterium during dehydrofluorinations in the presence of deuterium oxide, but no interconversions of pairs of stereoisomers were observed.

Thiol stimulation of the cytochrome p-450-dependent reduction of 1,1,1-trichloro-2,2-bis( p-chlorophenyl)ethane (DDT) to 1,1-dichloro-2,2-bis( p-chlorophenyl)ethane (DDD)

The enzymatic reduction of 1,1,1-trichloro-2,2-bis( p-chlorophenyl)ethane (DDT) to 1,1 dichloro-2,2-bis( p-chlorophenyl)ethane (DDD) was studied with rat hepatic microsomal fractions. The reaction required NADPH and was inhibited by dioxygen and carbon monoxide, which shows that the reaction is catalyzed by cytochromes P-450. Moreover, when the reaction was studied in the presence of deuterium oxide, no deuterium was incorporated into the DDD, which suggests that a one-electron reduction of DDT to the 1,1-dichloro-2,2-bis( p-chlorophenyl)ethyl radical followed by hydrogen atom abstraction accounts for the formation of DDD. The microsomal reduction of DDT to DDD was stimulated markedly by several thiols, including glutathione. The stimulatory effect of thiols was concentration dependent and was not due to conservation of cytochrome P-450, because nonthiol antioxidants failed to stimulate the reaction. The mechanism of the stimulation is not understood, but thiols do not promote the formation of DDD by preventing the alkylation of microsomal lipids and, thereby, stimulating the formation of a reduced alkane. Finally, these results show that soluble, lowmolecular weight compounds may enhance the activity of membrane-bound enzymes.

Deuterium oxide (D2O) enhances the photosensitivity of Stentor coeruleus

Stentor coeruleus exhibits negative phototaxis and step-up photophobic response (avoiding reaction) to visible light (maximum at 610-620 nm in both responses). In the presence of deuterium oxide (D2O) the step-up photophobic response was markedly enhanced, whereas the phototactic orientation response was inhibited. The induction time for the step-up photophobic response was longer in D2O than in H2O, and the duration of ciliary reversal for the response was also longer in D2O than in H2O, indicating that certain steps of the sensory transduction chain are subject to solvent deuterium isotope effects. The enhancement of the step-up photophobic response in D2O was canceled by LaCl3, while the inhibition of the phototactic orientation response in D2O was partially removed by LaCl3, even though LaCl3 did not affect the phototactic orientation response. These results suggest that the sensory transduction mechanisms for the two photoresponses are different, although the photoreceptors (stentorin) are the same.

ChemInform Abstract: PREPARATION OF 4‐(2‐DEUTERIO‐2‐PROPYL)ANISOLE THROUGH CATHODIC CLEAVAGE OF A SULFONE IN THE PRESENCE OF DEUTERIUM OXIDE

Chemischer InformationsdienstVolume 14, Issue 26 Preparative Organic Chemistry ChemInform Abstract: PREPARATION OF 4-(2-DEUTERIO-2-PROPYL)ANISOLE THROUGH CATHODIC CLEAVAGE OF A SULFONE IN THE PRESENCE OF DEUTERIUM OXIDE B. LAMM, B. LAMMSearch for more papers by this authorA. NILSSON, A. NILSSONSearch for more papers by this author B. LAMM, B. LAMMSearch for more papers by this authorA. NILSSON, A. NILSSONSearch for more papers by this author First published: June 28, 1983 https://doi.org/10.1002/chin.198326130AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume14, Issue26June 28, 1983 RelatedInformation

Preparation of 4-(2-Deuterio-2-propyl)anisole Through Cathodic Cleavage of a Sulfone in the Presence of Deuterium Oxide.

Preparation of 4-(2-Deuterio-2-propyl)anisole Through Cathodic Cleavage of a Sulfone in the Presence of Deuterium Oxide.

Evidence of Singlet Oxygen Participation in the Chlorophyll-Sensitized Photooxidation of Indoleacetic Acid

Chlorophyll-sensitized photooxidation of indoleacetic acid (IAA)-with chlorophyll extracted from Pisum sativum L. cv. Alaska W.R.-was determined in the presence of deuterium oxide and known quenchers of singlet oxygen ((1)O(2)) to explore the involvement of (1)O(2) in the reaction. O(2) uptake was measured in light in a buffered aqueous micellar system containing Triton X-100, KCl, chlorophyll, and IAA. The rate of O(2) uptake was zero in darkness. The reaction was stimulated by deuterium oxide and inhibited by sodium azide indicating that (1)O(2) participated in IAA photooxidation. Both mannitol and superoxide dismutase failed to inhibit O(2) uptake suggesting that neither the hydroxyl radical nor the superoxide anion played a significant role in the reaction.

Inactivation of normal and mutant Neurospora crassa conidia by visible light and near-UV: role of 1O2, carotenoid composition and sensitizer location.

Abstract— Inactivation of Neurospora crassa conidia from wild‐type and mutant strains by visible and near‐UV light has been investigated in the presence and absence of photosensitizing dyes. Inactivation by near‐UV is virtually unchanged by the presence of deuterium oxide or azide suggesting that, contrary to the situation with visible light and photosensitizing dyes, 1O2 is not involved in any substantial way in the formation of lethal lesions. The finding that carotenoid deficient strains are similar to wild‐type strains in sensitivity to near‐UV inactivation is consistent with 1O2 not being involved.Photodynamic inactivation of conidia by visible light occurs in the presence of methylene blue (MB), toluidine blue O (TB), or acridine orange (AO). Carotenoid deficient strains are more sensitive to such inactivation only when MB and TB are used. These results support the contention that MB and TB mediated damage involves the cell membrane where carotenoids are available for quenching, whereas AO mediated damage occurs in the nucleus sequestered from the protective influence of carotenoids.A newly isolated, lemon–yellow mutant, mapping to the al‐1 locus, exhibits sensitivities to photodynamic inactivation similar to other pure‐white mutants at the same locus. The sensitivity of this pigmented mutant is apparently related to insufficient unsaturation (seven to nine double bonds) of the two colored carotenoids, zeta–carotene and neurosporene, produced by the mutant.

Cyclic AMP in hypersensitivity reactions.

Phytohemagglutinin (PHA) was capable of inducing non-cytotoxic histamine release from human leucocytes. In the presence of deuterium oxide (D2O), PHA caused significantly greater histamine release. Dibutyryl cyclic AMP (d-cAMP) could enhance the histamine release in the presence of D2O although it was an inhibitor of the release if used alone. However, a beta agonist, isoproterenol, which increases intracellular level of cAMP was inhibitory with or without D2O. These data ask the question about dual effect of cAMP and suggest the possibility of different polls of cAMP in the target cells.

The role of microtubules in the response of macrophages to MIF

The role of the microtubular system in the response of macrophages to migration inhibitory factor (MIF) was investigated using agents which affect microtubule assembly. Microtubule-disrupting agents such as colchicine or vinblastine decrease or abolish the effect of MIF on macrophages. On the other hand, deuterium oxide enhances the effect of MIF on the macrophage. The presence of deuterium oxide during the first hour of interaction of MIF with macrophages is sufficient to increase the MIF response, whereas a pulse of D 2O during the first hour and subsequent addition of MIF had no effect. These results indicate that the state of the microtubule system during the initial period of macrophage-MIF interaction determines the response to MIF.

The degradative effects of porphyrins and heme compounds on components of the microsomal mixed function oxidase system.

The effects of in vitro treatment of the hepatic microsomal fraction with various porphyrin compounds on the activity and the content of the heme-containing components of the mixed function oxidase system were studied. The compounds examined were hematin, methemalbumin (with heme to protein molar ratio of 13:1 or 1:1), mesohemalbumin, bilirubin, biliverdin, mesoporphyrin IX, and protoprophyrin IX. The activity of the system was monitored by measuring its oxidative activity for the type I and type II substrates, ethylmorphine and aniline, respectively; as well as the microsomal contents of cytochrome P-450 and b5 and 14C-labeled heme, Mesoporphyrin IX was found to be most effective in inhibiting the oxidative activity of the mixed function oxidase system as well as in decreasing the microsomal contents of cytochromes P-450, b5, and heme. Biliverdin exerted no effect on these parameters. Hematin and the other compounds studied exerted variable inhibitory effects on the system. The degradative and inhibitory effects of protoporphvrin IX and mesoporphyrin IS could be blocked significantly by conducting the studies in the dark. The presence of biliverdin decreased the inhibitory effects of the porphyrins on the system; conversely the effects could be magnified in the presence of deuterium oxide. It is suggested that the mechanism by which porphyrins inhibit the mixed function oxidase system is through porphyrin-sensitized photo-oxidation of various constituents of the hepatic microsomal fraction and that the formation of singlet oxygen molecules is most likely involved in this process. Moreover the destructive effects of heme compounds on the microsomal components and activities of the drug-metabolizing mixed function oxidase system raise questions concerning the hypothesis that the components of this system, and specifically cytochrome P-450, are involved in the activity of the heme oxygenase system.

Electrochemical reactions. Part XVIII. Reductive cleavage of aromatic carbon–halogen bonds in the presence of deuterium oxide

Reductive dehalogenation of aromatic compounds at a mercury cathode in dimethylformamide containing 1% deuterium oxide with tetra-n-propylammonium perchlorate as supporting electrolyte may or may not result in deuterium incorporation, depending on the substrate. Compounds giving a radical anion intermediate which is detectable by cyclic voltammetry do not incorporate deuterium. The radical anion decomposes away from the electrode to a σ-radical which abstracts a hydrogen atom from the organic solvent. Compounds which give no detectable radical anion accept one electron in the initial step and fragment to a σ-radical at the electrode surface. This radical is reduced further to a carbanion which reacts with incorporation of deuterium into the product. Evidence for σ-radical intermediates in the latter cases comes from isolation of dimers or products of intramolecular radical cyclisation of appropriate substrates.

The stimulus-secretion coupling of glucose-induced insulin release. XII. Effects of diazoxide and gliclazide upon 45 calcium efflux from perifused islets.

The effect of diazoxide and gliclazide upon45calcium efflux from perifused islets was investigated. In the absence of extracellular calcium and presence of glucose, diazoxide caused an immediate and sustained increase in45calcium efflux. This facilitation of the outward transport of calcium might explain why diazoxide inhibits the stimulant action of glucose upon calcium accumulation in the beta cell. Under conditions known to abolish insulin release and the concomitant release of45calcium, namely in the absence of extracellular calcium or in the presence of deuterium oxide, gliclazide caused an immediate but transient decrease in45calcium efflux, this inhibition of the outward transport of calcium being observed both in the absence and presence of glucose and being followed by a significant rebound in45calcium efflux. It is suggested that this biphasic change in45calcium efflux could be due to a dual effect of gliclazide upon calcium handling by the beta cell, similar to that evoked by glucose and theophylline respectively.

The stimulus-secretion coupling of glucose-induced insulin release. IX. The participation of the beta cell web.

The rat pancreatic beta cell is surrounded by an ectoplasmic band of microfilamentous material located just beneath the plasma membrane. This cell web extends into the core of microvillous processes. Alteration of such a microfilamentous network by cytochalasin B (10 μg./ml.) results in a shortening or disappearance of the microvillous processes, a prominent margination of secretory granules and a redistribution of the cell web with the appearance of large and heterogenous masses of granular material extending into the cytoplasm. These ultrastructural changes are associated with a reversible enhancement of insulin release induced by glucose, theophylline or sulfonylurea in isolated islets of Langerhans. However, cytochalasin B fails to affect the basal rate of insulin release observed, respectively, at low glucose or calcium concentrations or in the presence of deuterium oxide. Cytochalasin B does not significantly modify the stimulant action of glucose on calcium accumulation and insulin biosynthesis in the isolated islets. These findings suggest that the microfilamentous cell web plays an important role in the active process of insulin secretion by controlling the access of insulin-containing secretory granules to the cell membrane.

Reactions of n-butene over a nickel exchanged 13X-type zeolite

The isomerization of n-butenes has been studied in a static system using as catalysts zeolites of the 13X-type with different extents of replacement of sodium ions by nickel ions. The presence of nickel enhances the catalytic activity considerably, comparable reaction rates with the nickel-containing zeolites being observed at temperatures 200 K lower than with the parent Na13X.A striking feature of the but-1-ene reaction was the preferential formation of trans-but-2-ene, which is compatible with a reaction involving radical intermediates. Further evidence in support of a radical, rather than a carbonium ion, mechanism was obtained by observing the isomerization (a), in the presence of deuterium (when an enhancement of rate and extensive exchange were observed) and (b), in the presence of deuterium oxide (when very little exchange occurred and the reaction rate was fractionally reduced).Detailed analysis of the exchange products in the reaction of but-1-ene and deuterium over the NiX catalysts, where butane was a major product, suggested that all three butenes exchange at rates which are comparable to the rates of isomerization, and that the hydrogenation process involves a very substantial redistribution of H and D atoms between the molecules.