Characterization of the early stages of programmed cell death in maize root cells by using comet assay and the combination of cell electrophoresis with annexin binding

An emerging topic in plant biology is whether plant cells display similar elements of programmed cell death (PCD) as animal cells do. We have studied cell death in maize roots exposed to cold stress by using fluorescence microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), DNA gel electrophoresis, single cell gel electrophoresis (SCGE), cell electrophoresis, and annexin binding techniques. The results showed that cell death in maize root cells triggered by cold stress was accompanied by a subset of features characteristic of animal PCD such as nuclear condensation and fragmentation, and oligonucleosomal DNA fragmentation. In addition to DNA laddering and TUNEL positivity, a "comet" pattern indicative of DNA breakage appeared as short as after one day of treatment. The maize root cell PCD process was also accompanied by an increase in negative surface charge of the dying cells due to exposure of phosphatiolylserine (PS) from inner to outer membrane. After annexin binding, however, the enhanced electrophoretic mobility (EPM) of the dying cells decreased nearly to normal values. This result suggests that the combination between cell electrophoresis and annexin binding provides a quantitative method for monitoring PS exposure during plant PCD.     

Estimation of the uncertainty of CRMs in accordance with GUM: Application to the certification of four enzyme CRMs

Uncertainties of four enzyme-CRMs that have recently been certified in a co-operation between the IRMM and the International Federation for Clinical Chemistry were estimated. Estimation was based on the sum of the uncertainties of characterization, homogeneity and stability. Data from the certification collaborative study were used to estimate laboratory uncertainties, which form the basis for the uncertainty of characterization. Estimations for the uncertainty of homogeneity were derived from classical homogeneity studies. The estimations of uncertainty of stability caused the most difficulties. Realistic uncertainties fitting the needs of customers while being derived from measurement data based on theoretical considerations were obtained. Received: 11 May 2000 / Revised: 21 June 2000 / Accepted: 27 June 2000     

Chemical Processes Involving 18-Crown-6-Ether in Activated Noncovalent Complexes with Protonated Peptides

These last decades, it has been widely assumed that 18-crown-6-ether (CE) plays a spectator role during the chemical processes occurring in isolated host-guest complexes between peptides or proteins and CE after activation in mass spectrometers. Our present experimental and theoretical results challenge this hypothesis by showing that CE can abstract a proton or a protonated molecule from protonated peptides after activation by collisions in argon or electron capture/transfer. Furthermore, thanks to comparison between experimental and calculated values of collision cross-sections, we demonstrate that CE can change binding site after electron transfer. We also propose detailed mechanisms for these processes.     

Continuous-flow fast atom bombardment mass spectrometry of oligonucleotides

Although frit-fast atom bombardment (frit-FAB) and continuous-flow FAB mass spectrometry have become standard methods for the analysis of peptides and peptide mixtures, these techniques have not been applied previously to the analysis of oligonucleotides. Mobilephase composition, flow rate, and sample size were optimized for the analysis of oligonucleotides by negative ion frit-FAB mass spectrometry (a type of continuous-flow FAB mass spectrometry). With a mobile phase consisting of methanol/water/triethanolamine (80:20:0.5, v/v/w), flow injection frit-FAB analysis of oligonucleotides showed lower limits of detection compared to standard probe FAB mass spectrometry. For example, in order to obtain a signal-to-noise ratio of 3:1, 38 prnol of d(GTIAAC) were required for frit-FAB mass spectrometry and 62 pmol were required for standard probe FAB mass spectrometry. The largest difference between frit-FAB and standard probe FAB was observed for d(pC)₅, for which the limit of detection by frit-FAB was approximately 11-fold lower than by standard FAB mass spectrometry. Adjustment of the mobile phase to pH 7 with trifluoroacetic acid increased the limit of detection (reduced sensitivity) a minimum of sixfold. Equimolar mixtures of two or three oligonucleotides produced deprotonated molecules in identical relative abundances whether analyzed by frit-FAB or standard probe FAB mass spectrometry. Finally, frit-FAB liquid chromatography mass spectrometry was demonstrated by separating mixtures of oligonucleotides on a β -cyclodextrin high-performance liquid chromatography column with a mobile phase containing methanol, water, and triethanolamine.     

Fast Field Echo imaging: an overview and contrast calculations

Current fast imaging techniques are based on gradient echo sequences with reduced flip angle excitation pulses and very short repetition times TR. Practical T2 values may be of the order of TR or longer. In this situation, a different image contrast can be obtained, depending on details of the sequence. Four essentially different versions of the basic Fast Field Echo (FFE) sequence can be distinguished and are described systematically in this article. For these sequences, image contrast formulas are presented. Practical imaging should tolerate small field inhomogeneities. This requirement can be satisfied by only three of the four versions. Numerical simulations are used to study the influence of a modified phase alternation scheme on image contrasts of two of the remaining sequences. The results of the calculations are verified by phantom studies on a 1.5-T whole-body imager. Implications for contrast in clinical images are discussed in relation to head images obtained on the same machine.     

EPR investigations of solid solutions of TSCC and TSCB doped with Mn2+

EPR investigations of Mn²⁺-doped solid solutions of TSCC and TSCB were performed between 4.2 K and 300 K. The Curie temperature vs. molar fraction of bromide x is nonlinear, for x ≧ 0,75 no ferroelectric EPR-line splitting occurs. The relation between the line widths and the molar fraction of bromide is discussed by means of a simple statistical model.     

A 15N labelling,FD and FAB study of ammonia loss from protonated arginine molecules

The positive ion field desorption (FD) spectrum of arginine taken at the best anode temperature only contains a peak due to [M+H]⁺ ions. At higher emitter temperatures a considerable amount of fragmentation is induced and the [M+H NH₃]⁺ ions become most abundant. Specific ¹⁵N labelling reveals that the eliminated ammonia molecule, exclusively, contains one of the terminal nitrogen atoms of the guanidyl group. This also applies to the ammonia loss from metastably decomposing [M+H]⁺ ions. The positive ion fast atom bombardment (FAB) spectrum shows more fragmentation than the FD spectrum. In contrast with the FD results, the [M+H]⁺ ions generated upon FAB with ion lifetimes <10⁻⁶ s eliminate both ammonia containing one of the terminal nitrogen atoms of the guanidyl group and ammonia containing the α-amino group in the ratio of 1.35, as found by ¹⁵N labelling. The metastably decomposing [M+H]⁺ ions, however, eliminate only the former ammonia molecule. In the negative ion FD and FAB spectra no other peak than that corresponding to the [M H]⁻ ion is observed. Some attention has been paid to the thermal degradation of arginine on the basis of a few Curie-point pyrolysis experiments.     

Observation of concentration boundary layers around growing or dissolving KH2PO4 crystals in stagnant solutions

The solute concentration profiles around KH₂PO₄ crystals growing or dissoloving in stagnant aqueous solutions were studied by the schlieren method after Toepler. By this technique the thicknesses of concentration boundary layers around the crystals, formed by free convection of solution, were measured in dependence of crystal size and super (under) saturation. In the case of dissolution, theoretical estimation of the boundary layer thickness yielded a fair agreement with the observed value. For growth, the process is dominantly limited by surface kinetics, whereas volume diffusion plays a minor role.