Structural analysis of monoclonal antibodies by ultrahigh resolution MALDI in-source decay FT-ICR mass spectrometry

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS. The method benefits from higher resolution of absorption mode FT mass spectra, compared to magnitude mode, which enables simultaneous identification of ISD fragments from both the heavy and light chains with a higher confidence in a wide mass range up to m/z 13 500. This method was applied to two standard mAbs, namely NIST mAb and trastuzumab, in preparation for method application in an interlaboratory study on mAbs structural analysis coordinated by the Consortium for Top-Down Proteomics. Extensive sequence coverage was obtained from the middle-down analysis (IdeS- and GingisKHAN-digested mAbs) that complemented the top-down analysis of intact mAbs. In addition, MALDI FT-ICR MS of IdeS-digested mAbs allowed isotopic-level profiling of proteoforms with regard to heavy chain N-glycosylation

A preliminary study in Wistar rats with enniatin : A contaminated feed

A 28-day repeated dose preliminary assay, using enniatin A naturally contaminated feed through microbial fermentation by a Fusarium tricinctum strain, was carried out employing two months-old female Wistar rats as in vivo experimental model. In order to simulate a physiological test of a toxic compound naturally produced by fungi, five treated animals were fed during twenty-eight days with fermented feed. As control group, five rats were fed with standard feed. At the 28th day, blood samples were collected for biochemical analysis and the gastrointestinal tract, liver and kidneys were removed from each rat for enniatin A detection and quantitation. Digesta were collected from stomach, duodenum, jejunum, ileum and colon. Enniatin A present in organs and in biological fluids was analyzed by liquid chromatography-diode array detector (LC-DAD) and confirmed by LC-mass spectrometry linear ion trap (MS-LIT); also several serum biochemical parameters and a histological analysis of the duodenal tract were performed. No adverse effects were found in any treated rat at the enniatin A concentration (20.91 mg/kg bw/day) tested during the 28-day experiment. Enniatin A quantitation in biological fluids ranged from 1.50 to 9.00 mg/kg, whereas in the gastrointestinal organs the enniatin A concentration ranged from 2.50 to 23.00 mg/kg. The high enniatin A concentration found in jejunum liquid and tissue points to them as an absorption area. Finally, two enniatin A degradation products were identified in duodenum, jejunum and colon content, probably produced by gut microflora

PER UN' ARCHEOLOGIA FORESTALE IN PIEMONTE. IL GIACIMENTO VILLAFRANCHIANO DELLA STURA DI LANZO (VILLANOVA NOLE CANAVESE).

Le indagini microxilotomiche condotte sui tronchi rinvenuti nella Stura di Lanzo nel sud del Piemonte, in terreni villafranchiani, hanno mostrato l'appartenenza di questi ad entità arboree della famiglia delle Taxodiaceae

Cytotoxic effects of the mycotoxin beauvericin to human cell lines of myeloid origin

Abstract Beauvericin, a cyclic hexadepsipeptide of potential importance to the health of humans and domestic animals, has been reported to exert cytotoxic effects on several mammalian cell types and to induce apoptosis.We investigated the cytotoxicity of this compound to two human cell lines of myeloid origin: the monocytic lymphoma cells U-937 and the promyelocytic leukemia cells HL-60. In some experiments HL-60 cells partially differentiated towards the eosinophilic phenotype were also used. Cultures of U-937 cells and HL-60 cells in stationary phase were exposed to beauvericin at concentrations ranging from 100nM to 300M for periods of time of 4 and 24 h, respectively. The effects of beauvericin on cell viability were assessed by the Trypan blue exclusion method. In another set of experiments, performed with U-937 cells, the mycotoxin was included in the culture medium at passaging, in order to assess its possible effects on cell growth. Viability of both U-937 cells and HL-60 cells was not affected by beauvericin at concentrations up to 3M, after 4 h exposure, whereas a steady decline was seen at higher concentrations. Similarly, after an exposure time of 24 h, a decline in viability was observed in cultures exposed to beauvericin at a concentration of 10M or higher. Thus, 50% cytotoxic concentrations at 24 h of¡«= 30M and¡«= 15M were estimated for U-937 cells and HL-60 cells, respectively. Similar experiments were performed with cultures of HL-60 cells partially differentiated towards the eosinophilic phenotype, revealing that, in 4 h exposure experiments (but not in 24 h experiments), the viability of these cultures underwent a significantly less pronounced decline, in comparison to undifferentiated HL-60 cultures. Interestingly, when U-937 cells were allowed to proliferate in the presence of the mycotoxin, included in the culture medium at passaging, a substantial cytotoxicity was observed at lower concentrations, compared with prevalently resting, stationary phase cultures. Accordingly, a definite inhibition of the proliferative capability of the cells was detected. The information provided by this work may be useful in selecting appropriate myeloid cell models for the development of biossays aimed at detecting beauvericin (and, possibly, other mycotoxins) in foods and other commodities. . 2003 Elsevier Ltd. All rights reserved