用4,4′-二羧酸-2,2′-二喹啉测定红细胞膜蛋白

细胞膜蛋白质的测定在生物物理、生物化学及医学等领域的研究中起重要作用,许多细胞膜上酶、离子、脂类、基团及功能的测定均与膜蛋白的水平有关,需测定膜蛋白量。目前膜蛋白的测定大多采用酚试剂法,该法灵敏度高,但酚试剂在碱性溶液中不稳定,干扰因素多,特异性差,在有非离子型表面活性剂存在下易形成难溶性沉淀,不能用于自动分析仪器。     

5,5′-联硫-2,2′-双硝基苯甲酸及其有关化合物对乙酰胆碱酯酶的别构效应

5,5'-联硫-2,2'-双硝基苯甲酸(DTNB)及其有关化合物TNB、TNB-Tch都是黄姑鱼肌肉乙酰胆碱酯酶的别构效应剂: 1.TNB非竞争性地抑制、而DTNB则激活该酶水解乙酰硫代胆碱和丁酰硫代胆碱的活力。对水解乙酰胆碱及α-萘酚乙酯的活力皆无影响。2.TNB-Tch比TNB具有更强的抑制作用。且对该酶水解乙酰胆碱、乙酰硫代胆碱及α-萘酚乙酯的活力皆有抑制作用。抑制类型与作用底物有关。底物在外周的结合能解除其抑制。3.钙离子可以解除TNB和TNB-Tch的抑制作用,而箭毒亦能与之对抗。对蛇毒乙酰胆碱酯酶,除了极低浓度(<10μM)DTNB和TNB有微弱的抑制作用外,TNB-Tch以及较高浓度的DTNB和TNB都具有强烈的激活作用。而对猪脑尾核乙酰胆碱酯酶的活力皆无影响。     

5,5′-联硫-2,2′-双硝基苯甲酸及其有关化合物对乙酰胆碱酯酶的别构效应

5,5'-联硫-2,2'-双硝基苯甲酸~*(DTNB)及其有关化合物TNB~(**)、TNB-Tch~(**)都是黄姑鱼肌肉乙酰胆碱酯酶的别构效应剂:1.TNB 非竞争性地抑制、而DTNB 则激活该酶水解乙酰硫代胆碱和丁酰硫代胆碱的活力。对水解乙酰胆碱及α-萘酚乙酯的活力皆无影响。2.TNB-Tch 比TNB 具有更强的抑制作用。且对该酶水解乙酰胆碱、乙酰硫代胆碱及α-萘酚乙酯的活力皆有抑制作用。抑制类型与作用底物有关。底物在外周的结合能解除其抑制。3.钙离子可以解除TNB 和TNB-Tch 的抑制作用,而箭毒亦能与之对抗。对蛇毒乙酰胆碱酯酶,除了极低浓度(<10 μM)DTNB 和TNB 有微弱的抑制作用外,TNB-Tch 以及较高浓度的DTNB 和TNB 都具有强烈的激活作用。而对猪脑尾核乙酰胆碱酯酶的活力皆无影响。     

Bronchial mucosal and kidney cortex affinity of 4- and 4,4′-substituted sulphur-containing derivatives of 2,2′-5,5′-tetrachlorobiphenyl in mice

In order to evaluate further the structural requirements previously proposed for accumulation of polychlorinated biphenyls (PCB) and their sulphur-containing metabolites in the respiratory tract of mice, 4-methylthio-, 4-methylsulphonyl and 4,4′-bis(methylthio)-2,2′,5,5′-[¹⁴C]tetrachlorobiphenyl were studied by whole body autoradiography. All the compounds gave rise to a strong accumulation of radioactivity in the mucosa of the bronchi, trachea and larynx. The first two substances were also concentrated in the mucosa of the nasal cavities. At the longer post-injection times all the compounds studied were localized in distinct sites of the kidney cortex. However, while the uptake of the monosubstituted sulphur-containing tetrachlorobiphenyl metabolites there was comparatively weak, the bis(methylthio) derivative showed a remarkable accumulation and retention in the kidney cortex. The study makes it possible to formulate the structural requirements for bronchial accumulation on the basis of the structure of the compounds that are accumulated rather than on the structure of the unmetabolized polychlorobiphenyls. Also with regard to the uptake in the kidney cortex a specific structure-dependency seems to exist.     

Synthesis of 2,2′-Anhydro-1 - (3′ -deoxy -3′ -iodo-β-D-arabino-furanosyl) thymine and Its Derivatives as Versatile Synthetic Intermediates

Abstract

2,2′ -Anhydro-1- (3′ -deoxy-3′ -iodo-5′ -O-trityl-B-D-arabinofuranosyl)-thymine (2) was synthesized from 2′,3′ -didehydro-3′-deoxythymidine (DHT) (1). Compound 2 was readily converted into 2′,3′-anhydro-lyxofuranosyl derivatives 4-6. Reaction of 4a with some nucleophiles (N₃ -, OMe-, Cl-) gave the corresponding 3′-substituted arabinonucleosides (7b,d,f) together with the minor xylosyl isomers (8a,c). Compounds 7b,d,f and 8a were deprotected to 7c,e,g and 8b, respectively.     

Oxidation of Morphine to 2,2′-Bimorphine by Cylindrocarpon didymum

The oxidation of morphine by whole-cell suspensions and cell extracts of Cylindrocarpon didymum gave rise to the formation of 2,2′-bimorphine. The identity of 2,2′-bimorphine was confirmed by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. C. didymum also displayed activity with the morphine analogs hydromorphone, 6-acetylmorphine, and dihydromorphine, but not codeine or diamorphine, suggesting that a phenolic group at C-3 is essential for activity.The morphine alkaloids are the major alkaloid components of opium, the dried latex material from cut seed capsules of the opium poppy, Papaver somniferum. Of all the alkaloids, the morphine alkaloid group has been studied in most detail, mainly due to the significant therapeutic properties these compounds possess. The morphine alkaloids are narcotic analgesics and are widely used by clinicians for the control of chronic pain. The use of microbial enzymes to provide biological routes for the synthesis of semisynthetic drugs that are difficult to synthesize chemically and as a means of producing new morphine alkaloid derivatives has been the subject of a significant amount of research; this topic has recently been reviewed (3). In recent years, there has been an increasing demand for new morphine alkaloid intermediates for the synthesis of novel semisynthetic drugs, and as part of a study to produce such compounds, we have been exploring fungal transformations of morphine. In this paper, we describe the conversion of morphine to pseudomorphine (2,2′-bimorphine) by Cylindrocarpon didymum 311186.

Biotransformation of morphine.

C. didymum 311186 was obtained from the International Mycological Institute (Egham, Surrey, United Kingdom). Mycelia were grown in media at pH 7.0 containing (grams per liter) yeast extract (10.0), KH₂PO₄ (10.0), (NH₄)₂SO₄ (5.0), and MgSO₄ (0.5). Trace elements were as described by Rosenberger and Elsden (9). Cultures were incubated at 30°C for 48 h with rotary shaking at 180 rpm. Washed mycelia (typically 0.5 g [wet weight]) were resuspended in 40 ml of medium containing 10 mM morphine (Macfarlan Smith Ltd., Edinburgh, United Kingdom) in 250-ml Erlenmeyer flasks. Samples (0.2 ml) were removed at regular intervals and diluted fivefold in 50 mM phosphoric acid (pH 3.5), to dissolve any insoluble metabolites. Mycelia were removed by centrifugation at 14,000 × g with an MSE Microcentaur microcentrifuge (Patterson Scientific Ltd., Dunstable, United Kingdom). The samples were analyzed by high-performance liquid chromatography (HPLC) with a Waters component system (Millipore Waters UK Ltd., Watford, United Kingdom). The HPLC system consisted of a 600E system controller connected to either a 484 absorbance detector or a model 994 programmable photodiode array detector set to 230 nm, 0 to 1 V full-scale detection. Injections of 50 μl were performed with a WISP 712 autoinjector and data processed with Millennium 2010 software. Separation of samples was achieved with a C₁₈ Spherisorb column (4.6 by 250 mm, 5-μm particle size; Anachem Ltd., Luton, United Kingdom), protected by a guard column of the same packing material with an isocratic solvent system containing 40 mM phosphoric acid buffer (pH 2.5) and acetonitrile in a ratio of 92:8 plus 2 mM pentanesulfonic acid, delivered at a flow rate of 1 ml/min. Analysis of the whole-cell incubation mixture by HPLC showed that morphine was completely removed from the medium after a period of 70 h. No other soluble metabolites were identified by HPLC; however, a white precipitate was found to accumulate in the incubation mixture. Microscopic analysis showed that the precipitate had formed regular cubic crystals. The crystalline material was found to dissolve under mildly acidic conditions, and HPLC analysis after such treatment revealed the stoichiometric conversion of morphine to an unknown compound (Fig. ​(Fig.1)1) that had a retention time that coincided with that of authentic 2,2′-bimorphine, kindly provided by M. McPherson (Macfarlan Smith Ltd.). The compound was analyzed by thin-layer chromatography (TLC) with polyester-backed plates precoated with Polygram Sil G/UV₂₅₄ (Machery-Nagel, Duren, Germany) and developed in ammonia-n-butanol (20/80 [vol/vol]). TLC analysis revealed the appearance of two compounds that were detectable under UV light at 254 nm and with Ludy Tenger reagent (7). Compound 1 had an R_f value of 0.42 corresponding to that of authentic morphine, while compound 2 had an R_f value of 0.25 which coincided with that of authentic 2,2′-bimorphine. 2,2′-Bimorphine shows greatly enhanced fluorescence characteristics, compared to those of morphine, due to extended conjugation (6). Compound 2 fluoresced with a characteristic blue color when the TLC plate was illuminated at 366 nm. Fluorescent excitation and emission spectra of compound 2 dissolved in 50 mM potassium phosphate buffer (pH 7.4) in 1-cm-path-length cuvettes were recorded with a Perkin-Elmer LS 50 B luminescence spectrometer (Perkin-Elmer Ltd., Beaconsfield, United Kingdom). Two principal excitation maxima were found at 280 and 320 nm, with a single emission maximum at 430 nm, typical of authentic 2,2′-bimorphine. Open in a separate windowFIG. 1Accumulation of 2,2′-bimorphine in whole-cell incubations of C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM morphine, and 0.5 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•) and 2,2′-bimorphine (○) concentrations were determined by HPLC. The data are means of three replicate incubations.

Identification of 2,2′-bimorphine.

¹H nuclear magnetic resonance spectroscopy of the product was performed at 400 MHz with a Bruker AM-400 spectrometer with tetramethylsilane as an internal standard and D-6 dimethyl sulfoxide as the solvent. The ¹H nuclear magnetic resonance spectrum gave the following signals, which were indistinguishable from those of an authentic sample of 2,2′-bimorphine (5) (for the proton assignments, see Fig. ​Fig.2,2, which gives the 2,2′-bimorphine numbering system): δ H 6.31 (2H, s, 1-H and 1′-H); 5.58 (2H, dd, J = 9.6 and 2.5, 7-H and 7′-H); 5.26 (2H, d, J = 9.6, 8-H and 8′-H); 4.70 (2H, d, J = 5.7, 5-H and 5′-H); 4.10 (2H, dd, J = 5.7 and 2.5, 6-H and 6′-H); 3.29 (2H, dd, J = 6.2 and 2.6, 9-H and 9′-H); 2.91 (2H, d, J = 18.6, 10β-H and 10β′-H); 2.57 (2H, d, J = 2.6, 14-H and 14′-H); 2.50 (2H, dd, J = 12.5 and 3.5, 16β-H and 16β′-H); 2.32 (6H, s, NMe and NMe′); 2.28 (2H, d, J = 12.5, α16-H and α16′-H); 2.23 (2H, dd, J = 18.6 and 6.2, α10-H and α10′-H); 1.99 (2H, dd, J = 11.4 and 3.5, α15-H and α15′-H); 1.68 (2H, d, J = 11.4, β15-H and β15′-H). Open in a separate windowFIG. 2Morphine analogs.The ¹H spectrum agreed with that expected for a symmetrical dimer, and only one aromatic proton signal was observed, instead of the characteristic AB pair of the morphine spectra, suggesting a symmetrical substitution on the aromatic ring. Laser desorption time-of-flight mass spectrometry was performed with a Kompact Maldi III mass spectrometer, and the mass spectrum showed a molecular ion, m/z 569.4, for C₃₄H₃₆N₂O₆.

Transformations of morphine analogs by C. didymum.

Whole-cell incubations of C. didymum were challenged with a range of morphine analogs including hydromorphone, 6-acetylmorphine, dihydromorphine, codeine, and diamorphine (see Fig. ​Fig.22 for structures). The incubations contained in 250-ml Erlenmeyer flasks approximately 0.61 g (wet weight) of mycelia and morphine analogs at 5 or 10 mM in a total volume of 40 ml of minimal medium. The flasks were incubated at 30°C with shaking, and samples were removed at intervals for HPLC analysis. Figure ​Figure33 shows that C. didymum was capable of activity with morphine, hydromorphone, 6-acetylmorphine, and dihydromorphine, and precipitates were observed to accumulate. Structural information on these products was not obtained. All of these compounds possess a free phenolic group at C-3 as a common structural feature which is likely to be an essential requirement for activity. This is consistent with the chemical oxidation of morphine to 2,2′-bimorphine, which requires the formation of a phenoxy radical intermediate (1). Open in a separate windowFIG. 3Transformations of morphine analogs by C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM substrate (5 mM dihydromorphine), and 0.61 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•), codeine (▵), diamorphine (▴), hydromorphone (○), dihydromorphine (□), and 6-acetylmorphine (■) concentrations were determined by HPLC.

Enzyme activity in cell extracts.

The whole-cell transformation of morphine to 2,2′-bimorphine prompted investigation of subcellular enzyme activity. Cell extract was prepared by the method of Rahim and Sih (8) with the following modifications. Frozen mycelia containing 10 to 14 g (wet weight) of biomass were placed in an ice-cold mortar with an equal weight of acid-washed white quartz sand (50/70 mesh; Sigma Chemical Company, Poole, United Kingdom) and an equal volume of ice-cold potassium phosphate buffer (pH 7.4). The mixture was ground with a pestle for approximately 20 min until it formed a thin paste. The paste was diluted with an equivalent volume of ice-cold buffer, and the sand and cell debris were removed by centrifugation at 20,000 × g for 15 min at 4°C in a Sorvall RC5C centrifuge fitted with an SS34 rotor. Protein was measured by the method of Bradford (2) with the Pierce protein assay reagent according to the manufacturer’s protocol. Typically, protein recoveries of approximately 7 mg of protein/g (wet weight) of cells were obtained. The fluorescent nature of 2,2′-bimorphine enabled the development of a convenient and sensitive enzyme assay. In reaction mixtures which contained potassium phosphate buffer (pH 7.4), morphine (5 mM), and cell extract, activity could be measured spectrofluorimetrically by measuring fluorescence of 2,2′-bimorphine at 440 nm when excited at 330 nm. Cell extract from mycelia harvested after 80 h of incubation with morphine had a specific activity of 0.36 U/mg of protein. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 2,2′-bimorphine from 2 μmol of morphine per min. No activity was observed in control reaction mixtures where the cell extract was replaced with boiled cell extract. Activity was inhibited completely when 0.1 mM azide was added to the reaction mixtures. Interestingly, no activity was observed in cell extract from mycelia that had not been incubated with morphine, which suggests that the activity is inducible. The development of a rapid and sensitive assay should facilitate the purification and characterization of the 2,2′-bimorphine-producing enzyme. 2,2′-Bimorphine has been shown to be a spontaneous reaction product of morphine in aqueous solutions, though the reaction was extremely slow (4). Furthermore, morphine can be oxidized to 2,2′-bimorphine with alkaline ferricyanide, a reaction which is known to proceed via a mesomeric aryloxy free radical, leading to the formation of the dimer (1). However, to the best of our knowledge, this is the first report of the microbial oxidation of morphine to 2,2′-bimorphine.     

3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).     

Synthesis of 2,2′-Anhydro Nucleosides with a Modified Sugar Side-Chain

Abstract

2,2′-Anhydro-1-(5,6-di-O-benzoyl-β-D-altrofuranosyl)thymine 6 and uracil derivative 7 are prepared by transformation of the corresponding 5′,6′-di-O-benzoyl-3′-O-mesyl-β-D-glucofuranosyl nucleosides 4 and 5 into the 2,2′-anhydro derivatives 6 and 7 using DBU.     

2,2′-Anhydro-4′-Thionucleosides: Precursors for 2′-Azido- and 2′-Chloro-4′-thionucleosides and for a Novel Thiolane to Thietane Rearrangement

Abstract

2,2′-Anhydro-4′-thio-β-and α-nucleosides 9 and 10 have been prepared by an in situ 4-thio-1,2-glycal addition route. They undergo ring-opening by azide or chloride ion to give, after deprotection, the 2′-substituted-4′-thionucleosides 13 and 14, whereas reactions with cyanide or fluoride sources lead to the unsaturated nucleosides 17 or 18, depending upon conditions. An unexpected and clean rearrangement to the thietane 23 occurs on treatment of uracil derivative 20 with DAST.     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by 4-acetoamido-4′-isothiocyanostilbene-2,2′-disulfonate

The permeabilities of sarcoplasmic reticulum vesicle membrane for various ions and neutral molecules were measured by following the change in light scattering intensity due to the osmotic volume change of the vesicles. 4-Acetoamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS), which is a potent inhibitor for the anion permeability of red blood cells membrane, inhibited the permeability of sarcoplasmic reticulum for anions such as Cl^?, P_i and methanesulfonate, while it slightly increased that for cations and neutral molecules such as Na⁺, K⁺, choline and glycerol. Binding of 5μmol SITS/g protein was necessary for the inhibition of anion permeability. These results suggest the existence of a similar anion transport system in sarcoplasmic reticulum membrane as revealed in red blood cell membrane.     

Synthesis of 2,2′-Anhydro-2-Hydroxy- and 6,2′-Anhydro-6-Hydroxy-1-β-D-Arabindfuranosylnicotinamide as Conformationally Restricted Nicotinamide Nucleoside Analogs1

Abstract

1-(β-D-Ribofuranosy1)-2(1H)-pyridone-3-carboxamide (6a) and the 6(1H)-pyridone derivative (6b) were prepared by condensation of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (3) with 2- and 6-hydroxynicotinic acid, respectively, to 4a and 4b, followed by conversion of the carboxylic acid function of 4a,b into their corresponding carboxamides 5, and then deprotection of 5. Bath 6a and 6b were then treated with 1,3-dichlom-1,1,3,3-tetraisopropyldisiloxane to give the corresponding 3′,5′-O-TPDS derivatives, 7a and 7b. Mesylation of 7a,b with mesyl chloride in pyridine afforded the stable, protected mesylates 8a,b. Upon de-O-silylation of 8a,b with ET₃NHF gave a mixture of unprotectd mesylates 9a,b and 2,2-anhydro- and 6,2′-anhydronucleosides, 1a and 1b. Upon storage of 9a,b at man temperature, they are quantitatively converted into 1a,b. Mild alkaline hydrolysis of 1a,b afforded their corresponding arabino nucleosides 10a,b.     

Anion recognition properties of 2,2′-bipyridine derivative receptors containing phenol group

A series of artificial 2,2′-bipyridine receptors (1, 2, 3) containing phenol group have been designed and synthesized. Their anion-binding properties are evaluated for various anions (F^?, Cl^?, Br^?, I^?, AcO^? and H₂PO₄^?) by UV-vis titration experiment in order to research the impact of different substituents on anion-recognition properties. Results indicate that the anion binding abilities can be tuned by electron push-pull properties of substituents on the phenyl ortho- or para-position of the receptors. In addition, receptor 1 is sensitive for F^? detection without the interference of other studied anions, and receptors 2 and 3 are sensitive for H₂PO₄^? detection.     

用固定化5′-磷酸二酯酶工业生产5′-核苷酸

自从固定化氨基酰化酶在日本获得工业应用以来,已引起各国广泛注意,至今已有近10个固定化酶在一些先进国家投入工业使用。我们自1970年开展固定化酶研究以来,已将我     

Rapid 2,2′-bicinchoninic-based xylanase assay compatible with high throughput screening**

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2-bicinchoninic-based protein reagent. Endo-1,4--d-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml^–1. The assay is linear for sugar concentrations from 0 to 86 g ml^–1 and can also be used to assay protein concentrations (0 to 143 g ml^–1) on the same plate. A variety of temperatures and pH conditions can be used and, after incubation, the assay requires only one detection reagent and one heating step.     

2,2′-Dithienyl diselenide pro-oxidant activity accounts for antibacterial and antifungal activities

The aim of this study was to explore if 2,2′-dithienyl diselenide (DTDS) pro-oxidant activity is related to its antibacterial and antifungal actions. The antimicrobial activity of DTDS against bacterial and fungal was investigated in the broth microdilution assay (3.02–387 μg/ml). Additionally, the survival curve of microorganisms in the presence of DTDS (12.09–193.5 μg/ml) was performed. The involvement of pro-oxidant activity in the DTDS antimicrobial action was investigated by supplementing the growth medium with 10 mM glutathione or ascorbic acid in the disk diffusion technique (0.64–640 μg DTDS/discs). The levels of reactive species (RS) induced by 25 mM DTDS were also determined. The results demonstrated that DTDS was effective in preventing the Gram-positive bacteria and Candida albicans growth. The minimum inhibitory concentration, twice and half concentrations of DTDS confirmed that the activity of compound was bactericidal for some microorganisms (Enterococcus faecalis, and Staphylococcus saprophyticus), bacteriostatic for Bacillus cereus and fungistatic for C. albicans. Antibacterial and antifungal actions of DTDS are related to the increase of reactive species levels. The presence of antioxidants in the growth medium avoided the DTDS antimicrobial action. In conclusion, DTDS showed promising antibacterial and antifungal actions, possibly related to its pro-oxidant activity.     

Studies of the interaction of tetramethylcucurbit[6]uril and 5,5′-dimethyl-2,2′-bipyridyl hydrochloride

The interaction between tetramethylcucurbit[6]uril (host) and 5,5'-dimethyl-2,2'-bipyridyl hydrochloride (guest) was studied by 1H NMR, X-ray crystallography, electronic absorption spectroscopy, fluorescence emission spectra and quantum chemistry calculations. This experimental-computational study that indicated the host can orientationally encapsulate the guest with a moderate association constant value. Computation qualitatively explained the split UV-visible absorption peak of the inclusion complex.     

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

Summary 2,2-dihydroxy-6,6-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.Dedicated to Prof. Dr. E. Ziegler on the occasion of his 70^th birthday     

Effect of Diquat (1,1′-Ethylene-2,2′-Dipyridylium Dibromide) on the Photosynthetic Growth of Rhodospirillum rubrum

Diquat (2 x 10(-4)m) inhibited both aerobic and anaerobic growth of Rhodospirillum rubrum. With photosynthetic cultures, diquat affected the synthesis of bacteriochlorophyll more readily than cell mass (turbidity). Diquat retarded the synthesis of bacteriochlorophyll and some protein more readily than that of other cellular constituents such as ribonucleic acid, deoxyribonucleic acid, and cell mass. With cells deficient in phosphate, diquat inhibited the uptake-conversion of inorganic phosphate completely only when 3-(3,4-dichlorophenyl)-1,1'-dimethyl urea and ascorbate were also present.     

2,2′-Dipyridyl diselenide is a better antioxidant than other disubstituted diaryl diselenides

The aim of this study was to investigate the in vitro antioxidant activity of 2,2'-dipyridyl diselenide (e) by comparing this effect with m-trifluoromethyl-diphenyl diselenide (a), p-fluor-diphenyl diselenide (b), p-chloro-diphenyl diselenide (c), and p-methoxyl-diphenyl diselenide (d) in rat liver homogenate. We also investigated if the mechanisms involved in the antioxidant property of 2,2'-dipyridyl diselenide are the same that of other diselenides. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC) levels were determined in rat liver homogenate, as indicators of antioxidant activity. Dehydroascorbate (DHA) reductase- and glutathione S-transferase (GST)-like activities, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities and the protection against the oxidation of Fe(2+) were determined to better understand the antioxidant property of compounds. δ-Aminolevulinic dehydratase (δ-ALA-D) activity was also carried out in rat liver homogenates, as a toxicological parameter. Compound e showed the highest potency in reducing TBARS (order of IC(50) values: e < b ≤ a < d ≤ c) and PC (order of IC(50) values: e < c ≤ b ≤ a < d) levels and lower potency in inhibiting δ-ALA-D activity than other diselenides. Compound e at all concentrations tested had no enzyme-mimetic property, but had radical-scavenging activity (≥5 μM) and protected against the oxidation of Fe(2+) (50 μM); while compounds a-d showed GST and DHA-mimetic activities and protected against the oxidation of Fe(2+), but had not radical-scavenging activities. This study indicates that (i) 2,2'-dipyridyl diselenide (e) had better in vitro antioxidant effect than other diselenides and lower inhibitory effect on δ-ALA-D activity, (ii) the presence of pyridine ring is responsible for the best antioxidant effect of this compound, and (iii) 2,2'-dipyridyl diselenide acts by different mechanisms of other diselenides.