Identification of Shiga toxins in Shiga toxin-producing Escherichia coli using immunoprecipitation and high-performance liquid chromatography-electrospray ionization mass spectrometry

We developed a rapid and reliable identification method for Shiga toxins in Shiga toxin-producing Escherichia coli (STEC) using immunoprecipitation and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Polyclonal antisera specific for Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) were raised in rabbits so as to be used for the immunoprecipitation. The immunoprecipitaion was carried out by mixing sample solutions with 50 microl each of the antisera to Stx1 and Stx2 followed by allowing the mixed solutions to stand for 30 min. The quantity required to obtain the immunoprecipitate was more than 0.5 microg of Shiga toxins. HPLC-ESI-MS analysis of the resulting immunoprecipitates provided accurate molecular weight information on Shiga toxins, leading to direct evidence for the presence of these toxins. It requires at most two days to perform our procedure from toxin extraction to measurement of HPLC-ESI-MS whereas the previous method using isolation procedures required about two weeks to complete. The usefulness of the present method has been demonstrated by identifying Stx1, Stx2 and a variant of Stx2 (Stx2e) in the immunoprecipitates prepared from STEC strains.     

Development of versatile cis- and trans-dicarbon-substituted chiral cyclopropane units: synthesis of (1S,2R)- and (1R,2R)-2-aminomethyl-1-(1H-imidazol-4-yl)cyclopropanes and their enantiomers as conformationally restricted analogues of histamine

The cyclopropane ring can be used effectively in restricting the conformation of biologically active compounds to improve activity and also to investigate bioactive conformations. We designed (1S,2R)- and (1R,2R)-2-aminomethyl-1-(1H-imidazol-4-yl)cyclopropanes (1 and 2, respectively) and their enantiomers (ent-1 and ent-2) as conformationally restricted analogues of histamine. The four types of chiral cyclopropanes bearing two differentially functionalized carbon substituents in a cis or trans relationship on a cyclopropane ring, (1S,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (7) and (1R,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (8) and their enantiomers (ent-7 and ent-8), were developed as the key intermediates for synthesizing 1, 2, ent-1, and ent-2. The reaction between (R)-epichlorohydrin [(R)-12] and phenylsulfonylacetonitrile (13a) in the presence of NaOEt in EtOH followed by treatment with acid gave the chiral cyclopropane lactone 11a with 98% ee in 82% yield. Compound 11a was converted into both the cis- and trans-chiral cyclopropane units 7 and 8, respectively, via reductive desulfonylation with Mg/MeOH as the key step. The corresponding enantiomers, the cis-substituted ent-7 and the trans-substituted ent-8, were also prepared starting from (S)-epichlorohydrin [(S)-12]. The four conformationally restricted target histamine analogues 1, 2, ent-1, and ent-2 were successfully synthesized from 7, 8, ent-7, and ent-8, respectively. The chiral cyclopropane units 7, 8, ent-7, and ent-8 should be useful as versatile intermediates for synthesizing various compounds having an asymmetric cyclopropane structure.     

Synthesis of potent beta-secretase inhibitors containing a hydroxyethylamine dipeptide isostere and their structure-activity relationship studies

Several beta-secretase inhibitors were designed based on hydroxyethylamine dipeptide isostere (HDI) structures and were synthesized by a methodology using the aza-Payne rearragement and O,N-acyl transfer reactions to study their structure-activity relationships. Among these pseudopeptides, effective compounds were developed as the first beta-secretase inhibitors containing the HDI transition state mimic with potent enzyme inhibitory activity (IC50 < 100 nM).     

Photoinduced electron-transfer systems consisting of electron-donating pyrenes or anthracenes and benzimidazolines for reductive transformation of carbonyl compounds

Photoinduced electron-transfer reactions of several ketone substrates were studied to evaluate the utilities of 1,6-bis(dimethylamino)pyrene (BDMAP), 1,6-dimethoxypyrene (DMP), 9,10-bis(dimethylamino)anthracene (BDMAA), and 9,10-dimethoxyanthracene (DMA) as electron-donating sensitizers cooperating with 2-aryl-1,3-dimethylbenzimidazolines. BDMAP and DMP generally led higher conversion of ketones and better yield of reduction products compared to BDMAA and DMA.     

The effect of humidity on thermal process of zinc acetate

The thermal decomposition of zinc acetate dihydrate Zn(CH₃CO₂)₂·2H₂O in some humidity-controlled atmospheres has been successfully investigated by novel thermal analyses, which are sample-controlled thermogravimetry (SCTG), thermogravimety combined with evolved gas analysis using mass spectrometry (TG–MS) and simultaneous measurement of differential scanning calorimetry and X-ray diffractometry (XRD–DSC). The thermal processes of anhydrous zinc acetate in dry gas atmosphere by conventional linear heating experiment initiated with the sublimation around 180 °C, followed by the fusion and the decomposition over 250 °C. SCTG was useful to interpret clearly the successive reaction because the high-temperature parallel decompositions were effectively inhibited. The thermal behavior changed dramatically by introducing water vapor in the atmosphere and the thermal process was quite different from that in dry gas atmosphere. Zinc oxide (ZnO) was formed only in a humidity-controlled atmosphere, and could be easily synthesized at temperatures below 300 °C. XRD–DSC equipped with a humidity generator revealed directly the crystalline change from Zn(CH₃CO₂)₂ to ZnO. A detailed thermal process of Zn(CH₃CO₂)₂·2H₂O and the effect of water vapor are discussed.     

Sulfation of hesperetin,naringenin and apigenin by the human cytosolic sulfotransferases: a comprehensive analysis

Abstract

Previous studies have revealed sulfation as a major pathway for the metabolism of hesperetin, naringenin and apigenin. The current study was designed to identify the human cytosolic sulfotransferase (SULT) enzyme(s) capable of sulfating these flavonoid compounds. Of the thirteen human SULTs, six (1A1, 1A2, 1A3, 1B2, 1C4, 1E1) displayed significant sulfating activity toward hesperetin, five (1A1, 1A2, 1A3, 1B2, 1C4) displayed sulfating activity towards naringenin, and four (1A1, 1A2, 1A3, 1C4) showed sulfating activity towards apigenin. Of the four human organ specimens tested, liver and intestine cytosols displayed much higher hesperetin-, naringenin- and apigenin-sulfating activity than lung and kidney cytosols. Moreover, sulfation of hesperetin, naringenin and apigenin was shown to take place in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under cultured conditions. Taken together, these results provided a biochemical basis underlying the metabolism of hesperetin, naringenin and apigenin through sulfation in humans.

     

A new synthetic route to 2-substituted naphtho[2,3-b]furan-4,9-dione2-Substituted Naphtho[2,3-b]furan-4,9-dione

4,9-Dimethoxynaphtho[2,3-b]furan 9 was obtained in 91% yield via the reductive methylation of naphtho[2,3-b]furan-4,9-dione 2 . After treatment of 9 with butyllithium, the mixture was allowed to react with N,N-dimethylacetamide, followed by oxidization with cerium(IV) diammonium nitrate to give 2-acetylnaphtho[2,3-b]furan-4,9-dione 1 . 2-Formylnaphtho[2,3-b]furan-4,9-dione 13 and 2-trimethylsilyl-naphtho[2,3-b]furan-4,9-dione 14 were also obtained from 9 by a similar method. The halodesilylations of 14 easily gave 2-iodonaphtho[2,3-b]furan-4,9-dione 16 , 2-bromonaphtho[2,3-b]furan-4,9-dione 17 , and 2-chloronaphtho[2,3-b]furan-4,9-dione 18 in 82%, and 93% and 83% yield, respectively. Furthermore, the nitrodesilylation of 14 gave 2-nitronaphtho[2,3-b]furan-4,9-dione 3 in 77% yield.     

Penetration of plasma surface modification. I. Cf4 and C2F4 glow discharge plasmas

Plasma treatment of a polymeric surface could involve at least three major mechanisms: (1) direct interaction of reactive species in the low-temperature plasma state with the surface (line of sight irradiation effect), and (2) chemical reactions of plasma-induced reactive species with the surface, and (3) reactions among reactive species and the surface (plasma polymerization). The first and the third effects are considered to be limited to the surfaces which directly contact with plasma (glow). The second effect is not limited to the surfaces that contact with plasma state but can penetrate beyond the plasma zone by diffusion. Using an assembly of fibers, of which only the top layer contacts with plasma (glow), the penetration of chemical changes caused by plasma exposure was investigated. Results indicate that the fluorination effect (incorporation of fluorine-containing moieties on the surface of polymeric substrate) penetrates through a considerable thickness of the assembly of fibers, depending on the porosity (gas permeability) of the system. Chemical reactions of plasma-induced (chemically) reactive but nonpolymerizing species with the substrate fibers seems to predominate. The direct interactions of energetic species, such as ions, electrons, and electronically excited species, with polymeric surfaces seems to play relatively minor roles in the plasma treatment investigated. The major role of plasma, in this case, seems to be creating such chemically reactive species. © 1994 John Wiley & Sons, Inc.