Energetics of MazG Unfolding in Correlation with Its Structural Features

MazG is a homodimeric α-helical protein that belongs to the superfamily of all-α NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-α NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual α-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH < 6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.     

Importance of Multiple Methylation Sites in Escherichia coli Chemotaxis

Bacteria navigate within inhomogeneous environments by temporally comparing concentrations of chemoeffectors over the course of a few seconds and biasing their rate of reorientations accordingly, thereby drifting towards more favorable conditions. This navigation requires a short-term memory achieved through the sequential methylations and demethylations of several specific glutamate residues on the chemotaxis receptors, which progressively adjusts the receptors’ activity to track the levels of stimulation encountered by the cell with a delay. Such adaptation also tunes the receptors’ sensitivity according to the background ligand concentration, enabling the cells to respond to fractional rather than absolute concentration changes, i.e. to perform logarithmic sensing. Despite the adaptation system being principally well understood, the need for a specific number of methylation sites remains relatively unclear. Here we systematically substituted the four glutamate residues of the Tar receptor of Escherichia coli by non-methylated alanine, creating a set of 16 modified receptors with a varying number of available methylation sites and explored the effect of these substitutions on the performance of the chemotaxis system. Alanine substitutions were found to desensitize the receptors, similarly but to a lesser extent than glutamate methylation, and to affect the methylation and demethylation rates of the remaining sites in a site-specific manner. Each substitution reduces the dynamic range of chemotaxis, by one order of magnitude on average. The substitution of up to two sites could be partly compensated by the adaptation system, but the full set of methylation sites was necessary to achieve efficient logarithmic sensing.     

Taurine biosynthesis enzyme cysteine sulfinate decarboxylase (CSD) from brain: The long and tricky trail to identification

Conclusion Most of the previous inconsistencies reported in the early works on CSD from brain, can be readily explained by the presence of two CSD activities in a brain extract in vitro. Their respective nature is now fully elucidated. On the one hand, the so-called CSD II activity is indeed a side activity expressed by GAD in vitro that is unlikely to play a physiologically relevant role in the biosynthesis of taurine in vivo. However it must be recalled that it represents the major contribution to the brain CSD total activity when measured in vitro. On the other hand, the so-called CSD I activity appears to be identical to liver CSD according to all biochemical evidence available to date. It is the most likely enzyme involved in taurine biosynthesis in vivo, and accordingly it represents a putative marker of taurine producing cells in the brain. It must be noticed that in the absence of specific inhibitors direct experimental evidence to support this hypothesis is still lacking. Taking into account all the data gathered in this review the CSD I and CSD II designation that referred only to a chromatography elution order has become obsolete and therefore must be henceforth abandoned. CSD II, as an activity expressed by GAD in vitro, must be called GAD associated CSD activity i.e. GAD/CSD., while CSD I as the brain enzyme identical to liver enzyme must be named CSD solely. According to our present immunocytochemical findings, this latter enzyme was not found in the cerebellum in nerve cells but in glial cells. These results provide the cellular basis for a role for taurine in relation to glial function, possibly as a general purpose regulator manufactured and released by glial cells.Special issue dedicated to Dr. Alan N. Davison.     

Présence d'un Gomphotheriidae indet. (Proboscidea, Mammalia) dans la faune vallésienne d'Afoud AF6 (Bassin d'Aït Kandoula, Maroc), établie d'après la microstructure de l'émail d'un fragment de molaire

The Aït Kandoula basin is one of the richest Miocene micromammals bearing areas known in the South of the High-Atlas, whereas the large mammal remains are scarce. The Afoud section in the center of the basin is well calibrated with the Geomagnetic Polarities Time Scale (KC95), and shows a succession of six fossiliferous layers. Beside rodents, the Afoud AF6 layer yields among some large mammal remains, a proboscidean identified as a Gomphotheriidae indet. from the enamel microstructure of a molar chip. This determination is grounded on the thickness of the enamel, on the prisms type, on the thinness and characteristics of the 3D zone and outside on the preferential course of the prisms.     

The laser in the Lowry technique for microdissection of freeze-dried tissue slices

Synopsis A new technique for tissue microdissection is described. This procedure, using an u.v.-laser micropreparation instrument, overcomes the extremely timeconsuming manual preparation. The u.v.-laser micropreparation design allows fast, precise, reproducible and smear-contamination-free tissue microdissection. The preparation of the tissue sample can be programmed by tracing out the area to be sampled with a non-destructive 0.5 m W He-Ne-laser aiming-beam. The trace is stored in a small electronic unit, which then guides the motor-driven stepping stage on the microscope in the actual dissection run with the u.v.-laser.The laser power is adjustable in the range 4 to 40 kW and controlled by a photo diode displayed on an oscilloscope screen. In the tissue slice, prepared according to Lowry, an unlimited number of cells or tissue compartments can be dissected and afterwards weighed. The procedure described offers a broader use of the quantitative microhistochemical techniques of Lowry and of Neuhoff.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September, 1975.