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Biomedical subjects

M Thellier

Publications and source records attributed to M Thellier.

At least 37 records · Page 2Linked to original sources

Cambium pre-activation in beech correlates with a strong temporary increase of calcium in cambium and phloem but not in xylem cells.

Using secondary ion mass spectrometry (SIMS), calcium was imaged in cambium cells and in the adjacent secondary phloem and xylem cells during the different phases of cambium functioning in beech (Fagus sylvatica L.). At the end of the period of quiescence, immediately before the resumption of cell divisions (i.e. at the cambium pre-activation phase), a strong temporary increase of calcium concentration was observed to take place in cambium and phloem but not in xylem cells.

Calcium↗

Imaging and microanalysis of 14N and 15N by SIMS microscopy in yeast and plant samples.

We have investigated the usefulness of Secondary Ion Mass Spectrometry (SIMS) for studying the tissue distribution of 15N labelling in yeast cells and soybean leaf tissues. The secondary ions best suited for this are 12C14N- and 12C15N-. Using a mass resolution of 6000, all problems of interference by other ions were avoided. The lateral resolution was of the order of 300 nm, i.e. well suited for subcellular studies. The sensitivity was good enough to allow the detection and the mapping of 15N, even when it was present at its natural value concentration of the isotopic ratio of only 0.37%. Using yeast cells at isotopic equilibrium with their nutrient medium, the nitrogen isotopic ratios in the cells were consistent with those in the medium. In the soybean leaf samples, the mapping of 14N and 15N was well correlated with the anatomical structures of the tissues. The mean isotopic ratios (100 15N/14N, at/at), measured in the leaf tissues by SIMS, were slightly below those in the nutrient medium as well as those measured in the leaf tissue by conventional mass spectrometry. This may be explained by differences in the methods of preparation of the leaf samples for SIMS and for mass spectrometry, and by the fact that the plants were probably still not perfectly at isotopic equilibrium with their external medium at the time the experiments were performed.

Cell Compartmentation↗

Polysaccharide distribution in the cellular junctions of immature fibre cells of flax seedlings.

The characteristic features of the pectins present in the walls of immature fibre cells of the hypocotyl of flax seedlings have been studied by a combination of three subtractive methods (treatment with boiling water, calcium chelator, and free endopolygalacturonase), three staining reactions (periodic acid-thiocarbohydrazide-silver, Ruthenium Red, and ferric hydroxylamine) and labelling with an endopolygalacturonase-gold probe. The primary wall and the periphery of the tricellular junctions were shown to contain pectic molecules made of blocks either with free acidic functions or methyl-esterified, these molecules being removed from the wall by splitting alpha (1-4) linkages. On the contrary, the pectic molecules in the core of the tricellular junctions were mainly with free acidic groups, but with an appreciable acetylesterification of their hydroxyl groups; and they were linked with one another chiefly by calcium bonds. This unexpected constitution of the core of the tricellular junctions may be considered to be an early marker of the cells destined to give rise to the fibre bundles of the mature plant.

Cell Wall↗

Kinetic study of the alpha-chymotrypsin-catalyzed hydrolysis and synthesis of a peptide bond in a monophasic aqueous/organic reaction medium.

We have studied the hydrolysis and synthesis reactions of the peptide bond involved in N-Cbz-L-tryptophanyl-glycineamide as catalyzed by alpha-chymotrypsin in various mixtures of water and 1,4-butanediol. Using a constant nonsaturating concentration of substrate, the initial reaction rates decreased exponentially with decreasing water content in the solvent mixture. When the water content was decreased from 100 to 20% (v/v), the maximum rate of reaction did not vary by more than a factor of 2 to 4, whereas the Michaelis constant increased exponentially. This exponential variation of the Michaelis constant was due to changes in the partitioning of the substrate between the active site of the enzyme and the solvent. In these processes, the actual rate constants did not vary when the relative contents of water and 1,4-butanediol were varied.

Animals↗

Determining the transient kinetic behavior of complex multi-enzyme systems by use of network thermodynamics.

As an example of the application of network thermodynamics to the treatment of complicated enzymatic systems, we have studied the transient kinetic behavior of a sequence of five enzymatic reactions, four with Michaelis-Menten kinetics and the final one with sigmoid kinetics. The object was to determine how the time-courses of the concentrations of all the intermediate substrates involved, depend on the effect of forward activation by the first substrate on the final enzymatic step. The case with forward activation exhibited an unexpected behavior with a reversal of the direction of the reaction before reaching equilibrium. The solution of the set of five non-linear differential equations was achieved using the student version of the simulation package PSPICE. The same approach can be utilized to study the behavior of any type of complex multienzymatic system (steady-states, transients, oscillations, chaos), or of combinations of enzymatic reactions with transmembrane transport in compartmental systems.

Multienzyme Complexes↗

The mapping of natural boron in histological sections of mouse tissues by the use of neutron-capture radiography.

Neutron-capture radiography has been applied to the mapping of natural boron in mouse histological sections. The method is based on the fact that the stable isotope boron-10 has an extremely large cross-section for the thermal-neutron reaction 10B(n, alpha)7Li. The local tissue boron concentrations were evaluated from the corresponding track densities of the nuclear reaction using an equation derived from a theoretical model of the system. The adjustable parameters of this equation were determined in two separate calibration experiments using standardized samples prepared by the addition of known amounts of boron. Interference from other nuclides engaged in nuclear reactions with thermal neutrons was also estimated. In the present experimental conditions the natural boron lower detection limit was 0.03 p.p.m. (fresh weight), and the spatial resolution was of the order of a few micrometers. Boron concentrations in mouse serum and urine were close to 0.22 and 0.57 micrograms ml-1, respectively. In the solid mouse tissues--liver, heart, brain, muscle and spleen--the concentration was usually low, ranging from 0.12 to 0.16 p.p.m. (fresh weight). They were significantly higher in the kidney, especially in the papilla (6.2 p.p.m. fresh weight). Apart from the kidney papilla, where boron was particularly concentrated in wall tubules, the boron distribution in most tissues appeared to be practically homogeneous. Natural boron, and even more so its enriched stable isotope 10B, appear as good candidates for molecular labelling using non-radioactive tracers.

Animals↗

Differential extractability of calcium and pectic substances in different wall regions of epicotyl cells in young flax plants.

We applied the simultaneous use of a subtractive method and two imaging techniques (secondary ion mass spectrometry and electron microscopy after PATAg staining) to correlate the distribution of Ca2+ to pectic substances in cell walls of young flax plants. The calcium images were compared with the structural electron microscopy images. This suggests that the linkage of the pectic substances within the wall is mainly by calcium bridges in the intercellular junctions of most types of cells under study (epidermis, subepidermis, fiber layer, and endodermis) and in the outer part (close to the cuticle) of the wall of the epidermal cells. In the primary walls of the various types of cells under study and in the inner part (close to the cytoplasm) of the wall of the epidermal cells, the linkage of the pectic substances would be mainly by covalent bonds. In the middle lamellae of the various cells, and in the intercellular junctions within the cortical parenchyma, both types of linkages apparently coexist. The mechanism of "ionic condensation" may provide an interpretation for the chemical status of the Ca2+ ions which are associated with the pectic components solubilized in boiling water, and which do not seem to contribute to the linkage of these components within the wall.

Calcium↗

Lithium transport in the mouse brain.

Using the stable isotopes of lithium 6Li and 7Li, and the nuclear reaction 6Li(n,alpha)3H for detection, we have studied the isotopic exchange of lithium in various areas of the mouse brain and in the mouse plasma, under conditions of constant concentration of total lithium. The neutron irradiations were performed using 'cold' neutrons, at the European Institute Von Laue-Langevin. The nuclear reaction track densities were determined using an automatic image analyser. In the plasma, the isotopic ratios, 6Li/7Li, were measured using 'Secondary Ion Mass Spectrometry'. The concentration of total lithium in the plasma was kept close to 0.28 mM. The brain concentration of total lithium (referred to the tissue water content) ranged from more than 2 mM in the thalamus to less than 0.65 mM in the white matter of the cerebellum. The Nernst potential of lithium thus ranged from approx. -50 to approx. -20 mV, which means that lithium is probably not far from electrochemical equilibrium between brain cells and plasma. At any moment, the isotopic abundance of 6Li (ratio of 6Li to total lithium) in the different brain areas, were not significantly different from one another. The time-course of the isotopic abundance of 6Li in the brain was fitted by the composition of two exponential terms. The time-course of the isotopic abundance of 6Li in the plasma was also fitted by the composition of two exponential terms. These analytic curves (for the brain and for the plasma) were not significantly different from each other, at the precision of the measurements. This means that the isotopic equilibration of lithium between brain and plasma is almost instantaneous (i.e. accomplished in a few min at the most).

Animals↗

Immunoreactivity of boronated antibodies.

Boronated antibodies have already been evaluated as agents in neutron capture therapy. Because the boronation procedure may alter the properties of the antibody it is important to study the immunoreactivity of the conjugated antibody before in vivo use. In our studies of two dextran-boronated monoclonal antibodies, anti-glial fibrillary acidic protein antibody, and anti-hyaluronectin antibody, we have used ELISA and immunohistological methods to determine antibody activity and specificity. A ten-fold decrease in activity was observed for both antibodies in ELISA, and non-specific interactions were seen in both immunohistological and ELISA procedures. The boron compound used was shown to be at least partly responsible for these non-specific interactions.

Animals↗

[The role of mineral ions in controlling morphogenesis in plants: a case of the inhibition of growth of the hypocotyle of Bidens pilosa L].

Mineral ions are implicated in various events occurring in the transduction of messages in plants, from the reception of the initial signal to the final morphogenetic expression. Ions may be involved also in the possible migration, storage and retrieval of the message. In a number of cases, cellular exchange of Ca2+ have been shown to occur at the reception of a signal. This is the reason why Ca2+ has often been considered as a "second messenger". For the possible message migration, cellular exchanger of Cl-, K+, H+ and Ca2+ are involved in the propagation of a wave of electric depolarization. The mechanisms underlying the possible storage of the message in a plant are still not clearly understood. However, ions such as K+, Na+ and Ca2+ interfere with the retrieval of the stored information. There are some indications that protons are involved in the metabolic reactions responsible for the final morphogenetic expression of the original signal. Moreover, the addition of Li+ ions inhibits, or shifts the latter effects, while the action of Li+ is counterbalanced by increasing the concentration of K+. The particular case of the inhibition of the growth of Bidens hypocotyles following delivering a few needle pricks to the cotyledons has been examined in more details.

Action Potentials↗

A method for the analysis of lithium in liquid droplets using isotopic dilution and SIMS measurements.

An absolute, fairly rapid method for the analysis of lithium in liquid droplets is described. It is based on the isotopic dilution of a 6Li (or 7Li)-enriched lithium salt, and on the measurement of isotopic ratios using Secondary Ion Mass Spectrometry. The sensitivity is well adapted to the analysis of lithium in minute volumes of plasma taken from patients suffering from manic-depressive psychosis.

Isotopes↗

How a soluble enzyme can be forced to work as a transport system: description of an experimental design.

The cellular transport systems which have been studied up to now have been found to be based on the functioning of specialized proteins anchored asymmetrically in cell membranes. In the present paper we show that a single soluble enzyme inserted at random in a gel slab can drive an uphill transport, provided that asymmetrical boundary conditions force the reversible reaction catalyzed by this enzyme to work forward on one face of the gel slab and backward on the other face. Experimentally, we have used a yeast alcohol dehydrogenase to induce an uphill transport of NADH. It cannot be excluded that comparable structurally symmetrical transport systems also exist in living cells. Such systems would be particularly well suited to preserving cell homeostasis with regard to small solutes.

Alcohol Dehydrogenase↗

Imaging the distribution of the stable isotopes of nitrogen 14N and 15N in biological samples by "secondary-ion emission microscopy".

Thanks to the "secondary-ion emission microscope" (CAMECA IMS 300), we have been able to image the distribution of the stable isotopes of nitrogen 14N and 15N in sections of plant roots (spatial resolution better than 1 micron), as well as to estimate the relative concentrations of these isotopes. The plants used (Lupinus spec.) originated from seeds with natural (i.e., 14N) nitrogen and had been fed for a few days with [15N]-nitrate before sampling. We have found in root sections of 6-day-old plants (prepared at 5 mm from the root tip) a clear-cut regionalization of the distribution of 15N between the vascular cylinder and the cortex. The latter contained approximately 5% 15N (of total nitrogen), whereas the relative concentration of the heavy isotope in the vascular cylinder was significantly lower. The observed concentration difference is probably due to the Casparian strip, which is a barrier for the apoplastic diffusion of solutes from the cortex to the vascular cylinder.

Mass Spectrometry↗

Microelectrode Measurements on Red Beet Vacuole : Biological Effect of Na OR NO(3) Ions, Diffusing from the Microelectrode.

Glass microelectrodes filled with 3 molar KCl are widely used to measure intracellular potentials and it is usual to try to minimize their electrolyte loss. In these experiments we have used the ionic leak of our microelectrodes, filled with various salt solutions, to introduce a given ion into the red beet vacuole. This allowed us to show that NO(3) (-) ions reduce the magnitude of the current spectral density while they do not change the resistance of the tonoplast. This is true when NO(3) (-) is either added to the external medium or used as the microelectrode filling solution. This can be interpreted by a NO(3) (-) effect on the vacuolar side of the tonoplast, resulting in an inhibition of the ion transporting ATPase. Replacing K(+) by Na(+) ions in the medium has no effect on tonoplast resistance (R(s)). On the contrary, when ions leaking from the microelectrode are H(+), Li(+) or K(+), R(s) is close to 4 kilohm square centimeter, whereas R(s) is of the order of 30KOmega square centimeter when Na(+) are the leaking ions. We also found a possible correlation between the presence of a Lorentzian in the current spectral density (cut-off frequency = 2 hertz) and a Cl(-) efflux from the vacuole. This could be explained by the existence of Cl(-) channels on the tonoplast.

Journal Article↗

Electrical noise measurements on red beet vacuoles : another way to detect the ATPase activity.

The vacuolar potential (V(vac)) and its fluctuations were recorded in red beet vacuoles (Beta vulgaris L.). Measurements with vacuoles in their suspension medium gave V(vac) = 10 +/- 2 millivolts (referred to the external medium) when 3 molar KCl microelectrodes were used. Buffering the microelectrode filling solution at pH 7.7 reversed the sign of the potential: V(vac) = -7 +/- 2 millivolts. The magnitude of the potential fluctuations was lowered by dilution (5-1000 times) with the suspension medium containing components released by the cells during the mechanical preparation. Fluctuations were decreased by 50 millimolar KNO(3) while they were enhanced by 5 millimolar ATP-Mg. No noticeable change in membrane resistance was detected. The presence of an ATPase bound to the tonoplast may explain the recorded noise spectra. These spectra imply a close connection between the rate of ATPase functioning and the magnitude of ionic fluxes across the tonoplast. It is suggested that noise analysis could be used to detect ATPase (or related enzyme) activity in vacuoles. Possible use of H(+) diffusion through a buffered microelectrode, to modify intravacuolar pH, is also suggested.

Journal Article↗

Lithium distribution in the brain of normal mice and of "quaking" dysmyelinating mutants.

Using nuclear reaction 6Li(n, alpha)3H and dielectric detectors, we have studied the distribution of Li in the brain of adult mice, following Li treatment of the animals. Two strains of animals were used in parallel: "quaking" dysmyelinating mutants and normally myelinated controls. The distribution appeared to be sharply regionalized in the brain of the normal mice (higher Li concentration in the gray rather than in the white matter, with the area postrema being particularly Li rich). In contrast, the Li distribution was practically homogeneous in the brain of the quaking dysmyelinating mutants, with a mean Li concentration comparable to that in the gray matter of the controls. The present method of Li detection has made it possible to estimate the Li equilibrium potentials (nerve cells with regard to plasma) in the different brain substructures. The results are consistent with (a) Li being actively extruded from nerve cells in all the cases and (b) myelination decreasing the relative importance of the passive component of Li transport in the nerve cells, as compared with the active component.

Animals↗