Background-Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.Methods-Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.Results-A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1-3(Fucα1-2)Galβ-containing carbohydrates, and . The lectin exerts antiproliferative activity specific to human leukemic T cells.
Conclusions-The protein belongs to the ricin B-like lectin superfamily, and has been designated as Clitocybe nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.General Significance-CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.
The distribution Pε of internal energies deposited in W(CO)6 (+•). ions upon charge stripping (that is, electron detachment to yield the doubly charged ion in the course of a single kiloelec-tronvolt energy collision) was estimated by a thermochemical method from the measured relative abundances of the doubly charged fragment ions produced. The thermochemical information needed to estimate P/ge was obtained by measuring the threshold translational energy losses associated with charge stripping of the singly charged fragment ions, W(CO) n (+) (n = 0-5). The P(/ge) curve falls exponentially with increasing internal energy. The average energy transferred to W(CO)6 (+•) upon a 7.8-keV collision with O2 is 19 eV, yielding W(CO)6 (2•) ions with an average of 4 eV of internal energy. In its general appearance, the P(ε) distribution associated with charge stripping is similar to the curves obtained from simple collisional activation of either W(CO) 6 (+•). or W(CO)6 (2+•) in kiloelectronvolt energy gaseous collisions. Given that charge stripping occurs by way of an electronic excitation process, this similarity in the energy deposition function is taken to indicate that electronic excitation is also the major mechanism for simple collisional activation in this system at zero scattering angle in the kiloelectronvolt energy regime. The internal energy distribution associated with a related charge-stripping process, charge inversion from the metal carbonyl anions to yield the corresponding cations, was also recorded. This reaction shows a large (∼7 eV) average internal energy deposition with a distribution that indicates near-zero probability of formation of unexcited ions. These data are tentatively interpreted in terms of vibrationalelectron detachment. The internal energy distribution associated with an exothermic process, charge exchange [W(CO)6 (2+•) + O2 → W(CO) + (6•)+O2 (+•)], was also characterized. Unexpectedly strong coupling of translational to internal energy is observed, and there is a large probability of depositing internal energies in excess of 10 eV, even though the exothermicity is only 3 eV. Finally, the internal energy distributions associated with the formation of doubly charged W(CO)6 (2+•) ions by electron ionization have been measured. Unlike the distribution for charge stripping, but like that for singly charged ions generated by electron impact, this distribution shows considerable structure, presumably due to Franck-Condon factors.
The mass spectrometric characterization of aqueous solutions of alpha- and beta-cyclodextrins (CDs) and o-, m- and p-coumaric acids (CAs) by negative ion electrospray ionization (ESI) indicates that the [CD+CA](-) ions were sourced from the inclusion complex present in solution and from the anion attached to CD molecules formed in the spray processes. The anion adducts formed in the spray process contribute significantly to the signal intensity of an ionized inclusion complex thus overestimating the calculated stability constant (K) of solution-phase complexes by one to two orders of magnitude. The relative intensities of anion adducts in mass spectra depend on the concentration ratio of the anion and the CD in spray droplets, while the relative intensity of the ionized inclusion complex depends on CD and CA concentrations in solutions and the value of K. Ion Mobility Spectrometry Mass Spectrometry [IMS-MS] measurements show that the collision cross-section (Omega) values of the [CD+CA](-) or [(CD)(2+)CA](2-) and [CD+CA](2) (2-) complex ions are 5-6% larger than or equal to CD(-) or [CD](2) (2-), respectively. Therefore, in the gas phase the anion adducts [CD+CA(-)] on cyclodextrin molecules possess the same conformations as the ionized inclusion complexes [CD+CA](-).
Ketoprofen (KP) is a nonsteroidal anti-inflammatory drug, which during UV irradiation rapidly transforms into benzophenone derivatives. Such transformation products may occur after topical application of KP, which is then exposed to sunlight resulting in a photo-allergic reaction. These reactions are mediated by the benzophenone moiety independently of the amount of allergen. The same reactions will also occur during wastewater or drinking water treatment albeit their effect in the aqueous environment is yet to be ascertained. In addition, only a few such transformation products have been recognised. To enable the detection and structural elucidation of the widest range of KP transformation products, this study applies complementary chromatographic and mass spectrometric techniques including gas chromatography coupled to single quadrupole or ion trap mass spectrometry and liquid chromatography hyphenated with quadrupole-time-of-flight mass spectrometry. Based on structural information gained in tandem and multiple MS experiments, and on highly accurate molecular mass measurements, chemical structures of 22 transformation products are proposed and used to construct an overall breakdown pathway. Among the identified transformation products all but two compounds retained the benzophenone moiety--a result, which raises important issues concerning the possible toxic synergistic effects of KP and its transformation products. These findings trigger further research into water treatment technologies that would limit their entrance into environmental or drinking waters.
A new analytical procedure using separation support based on Convective Interaction Media (CIM) was developed for speciation of Al in human serum at normal concentration levels. The separation of proteins was performed on a weak anion exchange CIM diethylamine monolithic column. Isocratic elution with buffer A (0.05 M tris(hydroxymethyl)aminomethane-hydrochloric acid + 0.03 M sodium hydrogen carbonate) was applied for 5 min, followed by linear gradient elution from 100% buffer A to 100% buffer B (buffer A + 1 M ammonium chloride) for the next 40 min. Separation of proteins was followed by UV detection at 278 nm. Separated Al species were detected online by inductively coupled plasma mass spectrometry. It was experimentally proven that 91 +/- 7% of Al in human serum was eluted under the transferrin peak. Transferrin was identified on the basis of the retention volume and by ACQUITY ultraperformance liquid chromatography-electrospray ionization mass spectrometry. The problem of extraneous contamination with Al was successfully overcome by using efficient cleaning procedures of eluents and chromatographic supports. The efficient cleaning was of paramount importance to perform Al speciation at extremely low concentration levels. The repeatability of measurement tested for six consecutive separations of unspiked serum was +/-8.6%. The limits of detection and quantification (based on 3 and 10 s of the blank) were 0.15 and 0.49 ng mL(-1) Al bound to transferrin, respectively. This is the first report on quantitative and reliable speciation of Al in human serum at normal concentration levels.
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