Immunoaffinity purification and characterization of 4-hydroxy-2-nonenal- and malondialdehyde-modified peptides by electrospray ionization tandem mass spectrometry

Two methods based on specific immunoaffinity enrichment followed by electrospray ionization (ESI) mass spectrometry (MS) have been developed for the specific analysis of 4-hydroxy-2-nonenal (HNE)- and malondialdehyde (MDA)-modified proteins (Michael and Schiff base adducts, respectively). Anti-HNE antibodies were immobilized on CNBr-activated sepharose, and the immunosorbent produced was used for the enrichment of HNE-adducted peptides originating from a model peptide modification and a tryptic digest of modified apomyoglobin. A further immunosorbent was produced by anti-dinitrophenyl immobilization and assayed for selective extraction of peptides modified with HNE and MDA that were initially converted to their respective hydrazones. Subsequent analysis and characterization of the different purified fractions by ESI-MS (MS/MS) revealed that the two immunosorbents enable efficient and specific enrichment of the carbonyl adducted proteins. This approach lowers substantially the detection limit of such modifications and, thus, enables better assessment and characterization of carbonyl modifications in biological and food systems.     

Sensitization to doxorubicin resistance in breast cancer cell lines by tamoxifen and megestrol acetate

Acquired drug resistance is a major factor in the failure of doxorubicin-based chemotherapy in breast cancer. We determined the ability of megestrol acetate and/or tamoxifen to reverse doxorubicin drug resistance in a doxorubicin-resistant breast cancer line (the human MCF-7/ADR). The cytotoxicity of doxorubicin, megestrol acetate, and/or tamoxifen was determined in the sensitive and resistant cell lines utilizing the sulphorhodamine B assay. Tamoxifen alone produced an IC50 (concentration resulting in 50% inhibition of control growth) of 10.6 microM, whereas megestrol acetate alone resulted in an IC50 of 48.7 microM in the MCF-7/ADR cell line. The IC50 of doxorubicin in MCF-7/ADR was 1.9 microM. Neither megestrol acetate alone nor tamoxifen alone at 1 or 5 microM altered the IC50 of doxorubicin. However, the combination of tamoxifen (1 or 5 microM) and megestrol acetate (1 or 5 microM) synergistically sensitized MCF-7/ADR cells. Additionally, megestrol acetate and tamoxifen inhibited iodoarylazidoprazosin binding to P-glycoprotein, and, in their presence, there was an increased doxorubicin accumulation in the MCF-7/ADR cells. Furthermore, the combination of tamoxifen and megestrol acetate had much less effect on the cytotoxicity of doxorubicin in MCF-7 wild-type cells. Clinically achievable concentrations of tamoxifen and megestrol acetate can largely sensitize MCF-7/ADR to doxorubicin. The combination of these three drugs in a clinical trial may be informative.     

Three-dimensional trajectory assessment of an IVUS transducer from single-plane cineangiograms: a phantom study

The recovery of the three-dimensional (3-D) path of the transducer used during an intravascular ultrasound (IVUS) examination is of primary importance to assess the exact 3-D shape of the vessel under study. Traditionally, the reconstruction is done by simply stacking the images during the pullback, or more recently using biplane angiography to recover the vessel curvature. In this paper, we explain, how single-plane angiography can be used with two projection models, to perform this task. Two types of projection geometry are analyzed: weak-perspective and full-perspective. In weak-perspective projection geometry, the catheter path can be reconstructed without prior transducer depth information. With full-perspective projection geometry, precise depth location of reference points are needed in order to minimize the error of the recovered transducer angle of incidence. The transducer angulation reconstruction is based on the foreshortening effect as seen from the X-ray images. By comparing the measured to the true transducer length, we are able to get its incidence angle. The transducer trajectory is reconstructed by stitching together the different estimated angulations obtained from each image in a cineangiogram sequence. The method is described and validated on two helical vessel phantoms, giving on average a reconstructed path that is less than 2 mm distant from the true path when using full-perspective projection.     

Simple identification of a cross-linked hemoglobin by tandem mass spectrometry in human serum

Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).     

A new fluorescence method for measuring cross-fluctuation fo two non-reactive components in a mixing device

A novel method for measuring local deviations from perfect mixing of two non-reative tracers is presented. It is based on the partially quenched fluorescence of one fluorescent tracer by a second tracer. When the concentration fluctuations are small, the deviation from the linear behaviour yields the cross-correlation of the fluctuating components of the concentrations of both traces from an – easy to record – average value of fluorescence intensity obtained in the focus of a laser beam. The method was proven to be suitable for measuring these quantities in a 1125 cm³ well-stirred tank as a function of location and stirring speed.     

Microbial mercury transformation in anoxic freshwater sediments under iron-reducing and other electron-accepting conditions

Potential rates of microbial methylation of inorganic mercury (added as HgCl2) and degradation of methyl mercury (MeHg) (added as CH3HgCl) were investigated in anoxic sediments from the Mobile Alabama River Basin (MARB) dominated by different terminal electron-accepting processes (TEAPs). Potential rates of methylation were comparable under methanogenic and sulfate-reducing conditions but suppressed under iron-reducing conditions, in slurries of freshwater wetland sediment In contrast, MeHg degradation rates were similar under all three TEAPs. Microbial Hg methylation and MeHg degradation were also investigated in surface sediment from three riverine sites, two of which had iron reduction and one sulfate reduction, as the dominant TEAP (as determined by 14C-acetate metabolism and other biogeochemical measurements). Methylation was active in sulfate-reducing sediments of a tributary creek and suppressed in iron-reducing, sandy sediments from the open river, whereas MeHg degradation was active at all three sites. Although iron-reducing conditions often suppressed methylation, some methylation activity was observed in two out of three replicates from iron-reducing sediments collected near a dam. Given that MeHg degradation was consistently observed under all TEAPs, our results suggest that the net flux of MeHg from iron-reducing surface sediments may be suppressed (due to inhibition of gross MeHg production) compared to sediments supporting other TEAPs.     

Involvement of Ligninlike Compounds in Toxicity of Dietary Alder Leaf Litter Against Mosquito Larvae

The toxicological characteristics of dietary decomposed alder leaf litter against mosquito larvae were further investigated through enzymatic and chemical purification of a phenoliclike cell-wall fraction isolated from crude litter. The toxicity of the subfractions obtained was controlled by standard bioassays on third instars of Aedes aegypti chosen as a reference target species. Enzymatic hydrolyses of the cell-wall fraction were performed with caylase, pectolyase, esterase, and -glycosidase, in order to release, respectively, cellulose material and phenolic compounds bound to lignins. These treatments did not affect the larvicidal activity and the phenolic activity of the cell-wall fraction. Chemical alkaline and acid hydrolyses were carried out to break ester and glycosidic bonds of the cell-wall fraction. Comparison of HPLC profiles of the hydrolysates from both toxic and nontoxic fractions did not reveal differences between the phenolic acids released. Aluminum chloride, known for its phenolic complexing activity, counteracted the larvicidal activity of the cell-wall fraction. Altogether, these results suggest the involvement of ligninlike compounds in the toxicity of dietary alder leaf litter against larval mosquitoes. The toxicity of this fraction, which was very sensitive to drastic and smooth oxidations, seemed to be associated with a strong oxidative potential. These results are discussed in relation to a possible mode of action of lignins in the plant–mosquito interactions.     

MACBETH — An Interactive Path Towards the Construction of Cardinal Value Functions

The application of classical models and techniques for cardinal measurement of values usually requires a person (D) to answer very difficult questions. The MACBETH approach, presented in this paper, proposes a simple questioning procedure to 'drive' the interactive quantification of values through pairwise verbal judgements of difference of attractiveness between valuable elements of a set A. Technically, MACBETH uses a chain of four linear programs for assigning numbers to the elements of set A based upon the qualitative judgements expressed by D and to reveal possible sources of incoherence. In practice, this information is to be used as a basis for discussion and learning. MACBETH stands for Measuring Attractiveness by a Categorical Based Evaluation TecHnique.     

Development of process-centered IPSEs in the ALF project

In this article we describe the main results of the ESPRIT project ALF (in French: Accueil de Logiciel Futur or Advanced Software Engineering Environments' Logistics Framework). In ALF we have developed a framework for Integrated Programming Support Environments (IPSEs), a software process modeling language (based on the notion of Models of Assisted Software Processes (MASP)), and an environment used to develop software process models. A MASP describes a software development method. This method specifies how an IPSE should behave. It can be plugged into the framework for IPSEs. Moreover, a MASP is used to integrate tools into the IPSE framework. In this article we focus on assistance and guidance facilities for software developers implemented in the framework for IPSEs.