Camille Ripoll

SIMS study of the calcium-deprivation step related to epidermal meristem production induced in flax by cold shock or radiation from a GSM telephone

ABSTRACT Exposing seedlings of the flax, Linum usitatissimum L., to a variety of weak environmental stresses plus a 2-day calcium deprivation triggers the common response of production of epidermal meristems in the hypocotyl. Here, we show that the same response was induced by a 1 min cold shock. Epidermal meristem production was also induced by a single 2-h exposure to radiation emitted at 0.9 GHz at non-thermal levels by a GSM telephone. This flax-based system is therefore well suited to studying the effects of low intensity stimuli, including those of electromagnetic radiation. To begin to determine the underlying mechanisms, in which calcium is implicated, it is desirable to analyse the changes in ions in the tissues affected. We therefore performed a Secondary Ion Mass Spectrometry (SIMS) study of the distribution of the main inorganic cations in the hypocotyl of control and calcium-deprived seedlings. This showed decreases in calcium, sodium and potassium and an increase in magnesium that did not alter substantially the overall ratio of divalent to monovalent cations.

Hypothesis: Hyperstructures regulate bacterial structure and the cell cycle

A myriad different constituents or elements (genes, proteins, lipids, ions, small molecules etc.) participate in numerous physico-chemical processes to create bacteria that can adapt to their environments to survive, grow and, via the cell cycle, reproduce. We explore the possibility that it is too difficult to explain cell cycle progression in terms of these elements and that an intermediate level of explanation is needed. This level is that of hyperstructures. A hyperstructure is large, has usually one particular function, and contains many elements. Non-equilibrium, or even dissipative, hyperstructures that, for example, assemble to transport and metabolize nutrients may comprise membrane domains of transporters plus cytoplasmic metabolons plus the genes that encode the hyperstructures enzymes. The processes involved in the putative formation of hyperstructures include: metabolite-induced changes to protein affinities that result in metabolon formation, lipid-organizing forces that result in lateral and transverse asymmetries, post-translational modifications, equilibration of water structures that may alter distributions of other molecules, transertion, ion currents, emission of electromagnetic radiation and long range mechanical vibrations. Equilibrium hyperstructures may also exist such as topological arrays of DNA in the form of cholesteric liquid crystals. We present here the beginning of a picture of the bacterial cell in which hyperstructures form to maximize efficiency and in which the properties of hyperstructures drive the cell cycle.

Physical methods to locate metal atoms in biological systems.

Publisher Summary Metal elements are involved in a number of cellular processes such as enzyme activation, transmembrane transport, water exchange, signal transduction, and intercellular communication. For instance, the alkaline and alkaline earth cations Na + , K + , Mg 2+ , and Ca 2+ play a key role in the functioning of living systems; Cu, Fe, Zn, or Mn may be associated with proteins; heavy metals and radioactive Sr 2+ or Cs + can be dangerous pollutants; and lithium or cobalt may be used as specific inhibitors of biological processes. Quantitatively mapping, or depth-profiling, metal atoms in biological systems can thus help toward an understanding of physiological mechanisms. A variety of physical methods are progressively being adapted to the study of biological specimens. This chapter discusses X-ray fluorescence, analytical electron microscopy (X-ray microanalysis, electron energy loss spectrometry, and Auger spectrometry), analysis of tracks originating from nuclear events, nuclear probes, and secondary ion imaging methods. Among these different methods, some can detect most chemical elements, whereas others apply to only a few elements. Some are useful mainly on a histological scale, whereas, in other cases, the spatial resolution may be good enough for considering subcellular localizations. In vivo measurements may be possible in a few cases, but most often, the methods are meant for fixed preparations. It is then clearly requisite that the techniques used for sample preparation—fixation, dehydration, and staining—do not disturb the natural distribution of the metal ions under observation.

A stochastic automaton shows how enzyme assemblies may contribute to metabolic efficiency

BackgroundThe advantages of grouping enzymes into metabolons and into higher order structures have long been debated. To quantify these advantages, we have developed a stochastic automaton that allows experiments to be performed in a virtual bacterium with both a membrane and a cytoplasm. We have investigated the general case of transport and metabolism as inspired by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) for glucose importation and by glycolysis.ResultsWe show that PTS and glycolytic metabolons can increase production of pyruvate eightfold at low concentrations of phosphoenolpyruvate. A fourfold increase in the numbers of enzyme EI led to a 40% increase in pyruvate production, similar to that observed in vivo in the presence of glucose. Although little improvement resulted from the assembly of metabolons into a hyperstructure, such assembly can generate gradients of metabolites and signaling molecules.Conclusionin silico experiments may be performed successfully using stochastic automata such as HSIM (Hyperstructure Simulator) to help answer fundamental questions in metabolism about the properties of molecular assemblies and to devise strategies to modify such assemblies for biotechnological ends.

Memory Processes in the Response of Plants to Environmental Signals

Plants are sensitive to stimuli from the environment (e.g. wind, rain, contact, pricking, wounding). They usually respond to such stimuli by metabolic or morphogenetic changes. Sometimes the information corresponding to a stimulus may be “stored” in the plant where it remains inactive until a second stimulus “recalls” this information and finally allows it to take effect. Two experimental systems have proved especially useful in unravelling the main features of these memory-like processes. In the system based on Bidens seedlings, an asymmetrical treatment (e.g. pricking, or gently rubbing one of the seedling cotyledons) causes the cotyledonary buds to grow asymmetrically after release of apical dominance by decapitation of the seedlings. This information may be stored within the seedlings, without taking effect, for at least two weeks; then the information may be recalled by subjecting the seedlings to a second, appropriate, treatment that permits transduction of the signal into the final response (differential growth of the buds). Whilst storage is an irreversible, all-or-nothing process, recall is sensitive to a number of factors, including the intensity of these factors, and can readily be enabled or disabled. In consequence, it is possible to recall the stored message several times successively. In the system based on flax seedlings, stimulation such as manipulation stimulus, drought, wind, cold shock and radiation from a GSM telephone or from a 105 GHz Gunn oscillator, has no apparent effect. If, however, the seedlings are subjected at the same time to transient calcium depletion, numerous epidermal meristems form in their hypocotyls. When the calcium depletion treatment is applied a few days after the mechanical treatment, the time taken for the meristems to appear is increased by a number of days exactly equal to that between the application of the mechanical treatment and the beginning of the calcium depletion treatment. This means that a meristem-production information corresponding to the stimulation treatment has been stored in the plants, without any apparent effect, until the calcium depletion treatment recalls this information to allow it to take effect. Gel electrophoresis has shown that a few protein spots are changed (pI shift, appearance or disappearance of a spot) as a consequence of the application of the treatments that store or recall a meristem-production signal in flax seedlings. A SIMS investigation has revealed that the pI shift of one of these spots is probably due to protein phosphorylation. Modifications of the proteome have also been observed in Arabidopsis seedlings subjected to stimuli such as cold shock or radiation from a GSM telephone.

Two‐dimensional electrophoresis investigation of short‐term response of flax seedlings to a cold shock

The flax, Linum usitatissimum L., is particularly suitable for studying the transduction and long‐term signal storage of environmental signals. To investigate the underlying molecular mechanisms, we have focused on the initial changes in the proteome since these offer the possibility of reflecting the plants history of exposure to stress. In principle, this ‘proteome signature’ might be revealed by two‐dimensional electrophoresis (2‐DE). We have therefore determined the potential of 2‐DE to study the kinetics of changes to the proteome of flax induced by a 1 min cold shock. Protein identification is difficult with flax because of the lack of knowledge of gene sequences. Nevertheless, 2‐DE analysis can be informative providing the significance of changes can be evaluated. We have developed a stringent threshold method to determine the significance of changes in gels obtained with proteins extracted from hypocotyls at different times after cold shock. This allowed us to reliably detect and characterize the kinetics of a set of seven spots that responded to cold shock and that constitute candidates for a proteome signature of long‐term signal storage.

Steady-state kinetic behaviour of functioning-dependent structures

A fundamental problem in biochemistry is that of the nature of the coordination between and within metabolic and signalling pathways. It is conceivable that this coordination might be assured by what we term functioning‐dependent structures (FDSs), namely those assemblies of proteins that associate with one another when performing tasks and that disassociate when no longer performing them. To investigate a role in coordination for FDSs, we have studied numerically the steady‐state kinetics of a model system of two sequential monomeric enzymes, E1 and E2. Our calculations show that such FDSs can display kinetic properties that the individual enzymes cannot. These include the full range of basic input/output characteristics found in electronic circuits such as linearity, invariance, pulsing and switching. Hence, FDSs can generate kinetics that might regulate and coordinate metabolism and signalling. Finally, we suggest that the occurrence of terms representative of the assembly and disassembly of FDSs in the classical expression of the density of entropy production are characteristic of living systems.

METHODOLOGICAL DEVELOPMENTS FOR APPLICATION TO THE STUDY OF PHYSIOLOGICAL BORON AND TO BORON NEUTRON CAPTURE THERAPY

The combination of immunogold labelling with electron microscopy or the direct detection of boron by electron energy loss spectrometry have the best lateral resolution for the imaging of boron or boron binding sites in tissues at the sub-cellular level. However these methods do not discriminate the boron isotopes. A number of physical methods make it possible to combine analytical imaging with isotopic labelling for boron studies in biological material. Secondary ion mass spectrometry has the potential to isotopically localise virtually any element with a resolution of ∼250nm with conventional instruments and 20–50nm with prototype instruments or with the NanoSIMS50; although SIMS has a relatively poor sensitivity for boron detection in biological matrices, boron imaging in plant samples is possible. Laser microprobe mass analysis also has the potential to detect boron isotopes with a lateral resolution of 3 to 5 μm and a detection limit of a few tens of μg/g with the conventional instruments and of the order of 1ng/g with the new LARIMP system; although mass resolution of LMMS is in general not very good, the risk of interference by other ions at the level of boron masses is limited. Neutron capture radiography is probably the easiest technique for boron imaging and boron isotopic labelling studies in tissues and sometimes at the sub-cellular level, although it detects only 10B isotopes. Nuclear reactions with charged particles (nuclear reaction analysis) have the potential to detect both isotopes of boron and carry out absolute boron concentration measurements with minimal matrix effects, limited risk of interference by other nuclides, a lateral resolution of a few μm at the best, a detection limit better than 1 μg/g for 11B, of the order of 10 μg/g for 10B and an accuracy of 1 to 2% in the determination of 10B/11B isotopic ratios. Preventing the diffusion of possibly mobile forms of boron during the preparation of the biological specimens is still a difficult problem for most techniques. The appropriate application of those methods, or their mutual combination or combination with other methods has made it possible: i) to yield information about the boron concentrations and fluxes in sub-cellular compartments and support the view that the cellular transport of boron was mainly passive under the experimental conditions under consideration; ii) to image the distribution of boron and of boron binding sites in tissues and sometimes at the sub-cellular level; iii) to study the short-distance diffusion and the long-distance transport of boron in plants and to assess the role of the phloem in the long-distance transport in various plant species; iv) to determine the origin (seed reserves vs uptake by roots) of the boron present in different sub-cellular compartments. For boron neutron capture therapy of cancers, invasive techniques of boron detection and imaging are comparable to the techniques described above for the study of physiological boron; for clinical applications, non-invasive techniques to follow 10B-compounds in vivo are being developed, especially by targeting of such compound by 18F and the use of positron emission tomography or by direct detection of 10B by magnetic resonance imaging.

Combed Single DNA Molecules Imaged by Secondary Ion Mass Spectrometry

Studies of replication, recombination, and rearrangements at the level of individual molecules of DNA are often limited by problems of resolution or of perturbations caused by the modifications that are needed for imaging. The Combing-Imaging by Secondary Ion Mass Spectrometry (SIMS) (CIS) method helps solve these problems by combining DNA combing, cesium flooding, and quantitative imaging via the NanoSIMS 50. We show here that CIS can reveal, on the 50 nm scale, individual DNA fibers labeled with different, nonradioactive isotopes and, moreover, that it can quantify these isotopes so as to detect and measure the length of one or more short nucleic acid fragments associated with a longer fiber.

Shell microlaminations of the freshwater bivalveHyridella depressa as an archival monitor of manganese water concentration: Experimental investigation by depth profiling using secondary ion mass spectrometry (SIMS)

Specimens of the freshwater unionid bivalveHyridella depressa were experimentally exposed to a synthetic river water containing an elevated Mn water concentration (20 mg l−1) for 2 or 6 days. SIMS depth profiles through the incremental nacre microlaminations or tablets (∼0.6 μm breadth) of the shells of these bivalves showed increases in the signal intensity of Ca-normalised Mn that corresponded to the period of exposure. These results support the proposition that bivalve shells can be used as retrospective monitors of water chemistry. They also indicate that 1) there is a lag phase between exposure to the elevated Mn water concentration and its expression in the shell, and 2) the period for Mn in the shell to reach equilibrium with the aquatic medium is greater that 2 to 6 days.