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.     

Preparation of Hydrophobically Modified Poly(amidoamine) Dendrimer-Encapsulated Gold Nanoparticles in Organic Solvents

HAuCl(4) in aqueous solution was extracted to toluene or chloroform using a hydrophobically modified poly(amidoamine) dendrimer. Then, by reduction of Au(3+) ions with dimethylamineborane, gold nanoparticles in the size range of 2-4 nm were obtained in toluene or chloroform. It is suggested that gold nanoparticles are encapsulated by the dendrimer. Copyright 2000 Academic Press.     

Kinetic stabilization of the [1.1]paracyclophane system: isolation and X-ray structural analysis of a [1.1]paracyclophane derivative and its interconversion with the transannular adduct

Intentionally designed kinetic stabilization of the [1.1]paracyclophane skeleton has been achieved by multiple substitution of the aromatic rings with trimethylsilylmethyl and N,N-dimethyl-carbamoyl groups, which serve to shield the proximate bridgehead carbon atoms sterically from access by other reagents. The bis(Dewar benzene) precursor (1a) has been prepared in essentially the same manner as previous derivatives--starting from the photocycloaddition of 1,4-bis(trimethylsilyl)-2-butyne to octa-hydroindacene-1,5-dione--except for a few critical modifications described in the text. Substituted [1.1]paracyclophane (2a), photochemically generated from the precursor, is indefinitely stable at 50 degrees C and suffers decomposition only by 8 % after 2 h at 100 degrees C in degassed n-decane, demonstrating its greatly improved kinetic stability compared to previous [1.1]paracyclophanes. Since 2a undergoes efficient photochemical transformation into the transannular addition product 3a, irradiation of la tends to produce a mixture of products consisting mainly of 3a. Compound 3a, however, reverts thermally to 2a in a process of half life 40 min at 55 degrees C; the activation parameters for this process are deltaH(not equal to)= 21.1 +/- 0.8 kcalmol(-1) and deltaS(not equal to) = -10.5 +/- 2.6 cal K(-1)mol(-1). Thus, on heating 3a in benzene and cooling the resultant solution, 2a is obtained as orange-red crystals. X-ray crystallographic analysis of 2a reveals benzene rings bent to the highest degree ever reported for a paracyclophane, with their face-to-face arrangement in unusually close proximity. The shortest nonbonding interatomic distance is 2.376 A; less than the sum of the van der Waals radii by more than 1.0A. The generation of related substituted [1.1]paracyclophanes and their kinetic stabilities are also reported.     

Leaching behavior of metal-containing species in residual oils with different organic solvents

The leaching behavior of two different residual oils is investigated with 24 organic solvents including alkanes, aromatics, esters, ethers ketones, alcohols and nitrogen compounds. A 2-g portion of sample oil is shaken with 20 ml of the organic solvent. The leaching of the organic matrix of the oil is determined by weighing the dissolved organic matrix after removal of solvent. The leaching of vanadium, iron and nickel was determined by flame atomic absorption spectrophotometry after ashing of the dissolved fraction. The leaching of those metals depends strongly on the solvent used; for example, vanadine is almost completely leached into toluene, but only slightly into methanol. The leaching of iron is poor compared with that of vanadium or nickel. In most solvents, leaching of those metal-containing species is poorer than that of organic material. Exceptionally, N,N-dimethylformamide is more efficient in leaching vanadium species than organic material.     

Spectroscopic investigation of cyclodextrin monomers,derivatives, polymers and azo dyes (1)

Circular dichroism (CD) and visible spectra of inclusion compounds between Methyl Orange (MO) analogues and -, -, -cyclodextrin (cdx), 2,6-dimethyl-and 2,3,6-trimethyl--cdx, water soluble -, -, -cdx polymer products were investigated. In the CD-spectroscopic investigation, the complex with -cdx epichlorohydrin condensate showed a large amplitude and splitting of the induced ^* band. Fractions of glyceryl ether of less than 2000 and polymer of more than 10000 dalton molecular mass were separated. Complexes of above two fractions and MO showed the same splitting spectral pattern. Job's plots from visible spectra showed the formation of the 11 complex and CD-data suggested the co-existence of the 21 MO-cdx complex. This splitting pattern showed the reversal of the signs when -cdx-ethyleneglycol-bis(epoxypropyl) ether was used and disappeared when larger host molecules and azo dyes were used. The splitting was explained by exciton interaction.     

Involvement of human small fragment nuclease in the resistance of human cells to UV-C-induced cell death

Human small fragment nuclease (Sfn) is one of the cellular proteins that were reported to degrade small, single-stranded DNA and RNA. However, the biological role of Sfn in cellular response to various stressors such as UV-C (mainly 254 nm wavelength ultraviolet ray) remains unclear. We have examined whether modulation of human SFN gene expression affects cell survival capacity against UV-C-induced cell death, analyzing colony survival ability in UV-C-sensitive human RSa cells treated with short double-stranded RNA (siRNA) specific for SFN messenger RNA (mRNA). The expression levels of SFN mRNA in the siRNA-treated RSa cells decreased to about 15% compared with those in the control siRNA-treated cells. The siRNA-treated RSa cells showed lower colony survival and higher activity of caspase-3 after UV-C irradiation than the control siRNA-treated RSa cells. Furthermore, the removal capacity of cyclobutane pyrimidine dimers (CPD) in the siRNA-treated RSa cells decreased compared with the control siRNA-treated RSa cells. There was no difference in the colony survival and CPD removal capacity after UV-C irradiation between the control siRNA-treated RSa cells and mock-treated RSa cells. These results suggest that SFN expression is involved in resistance of RSa cells to UV-C-induced cell death through the roles it plays in the DNA repair process.     

Convenient indole synthesis from 2-iodoanilines and terminal alkynes by the sequential Sonogashira reaction and the cyclization reaction promoted by tetrabutylammonium fluoride (TBAF)

The sequential Sonogashira reaction and the cyclization reaction of various 2-iodoanilines and terminal alkynes in the presence of a palladium catalyst and tetrabutylammonium fluoride (TBAF) gave the corresponding 2-substituted indoles in good yields.     

Selective foam separation of binary protein solution by SDS complexation method

A fundamental study about the selective foam separation of protein mixture was carried out. A solution containing two proteins, ovalbumin (OA) and lysozyme (LZ), and an anionic surfactant, sodium dodecyl sulfate (SDS), was adjusted to pH 6.0, which referred to an intermediate state between the isoelectric points of the proteins. The solution was processed by continuous foam separation. The results showed that a proper addition of SDS greatly improved the selective recovery of LZ to OA. The experimental data were well explained by a simple model that most of cationic protein molecules (LZ) are associated with SDS and the adsorption of all the species including LZ-SDS complexes are subjected to Langmuir adsorption isotherm. The results also showed that one of the Langmuir parameters, which means a kind of lyophillic property of adsorbed material, of LZ-SDS complexes was extremely large as compared with that of primary protein.     

On the inverse Sturm-Liouville problem for spatially symmetric operators,II

Using a third order Picard-Fuchs equation we show that a certain two parameter family of planar vectorfields for parameter values in a certain cone has a unique limit cycle, which is born from a Hopf bifurcation and dies in a saddle connection. This removes a superfluous hypothesis in Theorem 3.2, Chapter 13 of S. N. Chow and J. K. Hale (“Methods of Bifurcation Theory,” Springer-Verlag, New York, 1982).