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Biomedical subjects

S F Mathias

Publications and source records attributed to S F Mathias.

6 recordsLinked to original sources
PubMed1993

Plant viral leaders influence expression of a reporter gene in tobacco.

In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter beta-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two- to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four- to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four- to six-fold more efficiently than the random leader control.

Base Sequence↗
PubMed1990

Water, temperature and life.

Cold is the fiercest and most widespread enemy of life on earth. Natural cold adaptation and survival are discussed in terms of physicochemical and biochemical water management mechanisms, relying on thermodynamic or kinetic stabilization. Distinctions are drawn between general effects of low temperature (chill) and specific effects of freezing. Freeze tolerance is a misnomer because tolerance does not extend to the cell fluids. Freezing is confined to the extracellular spaces where it acts as a means of protecting the cytoplasm against freezing injury. Freeze resistance depends on the phenomenon of undercooling, a survival mechanism that relies on the long-term maintenance of a thermodynamically highly unstable state. Correct water management involves many factors, among them the control of membrane composition and transmembrane osmotic equilibrium, the biosynthesis of compounds able to afford protection against injury through freeze desiccation and the availability (or inactivation) of biogenic ice nucleation catalysts.

Adaptation, Physiological↗
PubMed1985

Preservation of viable cells in the undercooled state.

Previous studies into the mechanisms governing the freezing of cells in the absence of extracellular ice have been extended to develop a method for the preservation of viable cells in the undercooled state. Deep undercooling of cells is achieved by suspending fine droplets of the cells in oil to make an emulsion, thus minimizing initiation of extracellular ice nucleation. Attempts to preserve yeast cells, cultured sainfoin cells, and dissected shoot-tips (pea and potato) in this way are described. The main findings are that yeast cells can be preserved undercooled at -20 degrees C for at least 16 weeks with no detectable loss of viability, showing that -20 degrees C is a low enough temperature for inhibition of significant biochemical deterioration and that the emulsions are stable over long periods. In preliminary experiments, sainfoin cells survived 24 hr at -10 degrees C, and shoot-tips survived 48 hr at -10 degrees C. Sainfoin cells, conditioned by growth in medium supplemented with sorbitol, showed enhanced survival after exposure to low temperatures and a lower intracellular freezing point than control cells. Possible reasons for this are discussed.

Animals↗
PubMed1984

Nucleation and growth of ice in deeply undercooled erythrocytes.

Previous studies of the mechanism of freezing of erythrocytes in the absence of intracellular ice have been extended to define the catalytic sites responsible for promoting nucleation. The following aspects have been investigated: (1) the freeze propagation between undercooled erythrocytes, (2) the nucleation of ice in undercooled erythrocyte ghosts, and (3) the freezing behavior of undercooled hemoglobin solutions. The main findings are: (1) no cross-nucleation occurs between individual cells packed within the same emulsified water droplet; (2) the differential scanning calorimetric power-time curves of intact cells and ghosts are identical, indicating that hemoglobin does not affect ice nucleation; (3) the nucleation temperature of ice in an aqueous solution of hemoglobin (isolated from the cells) is substantially lower than that for the same solution when contained in the intact cell; (4) the threefold freeze concentration which accompanies the freezing of a 25% hemoglobin solution does not cause denaturation of the protein.

Blood Preservation↗
PubMed1983

Ice nucleation and freezing in undercooled cells.

DSC has been employed to study the effect of cooling on a range of cells under exclusion of extracellular ice and in the absence of chemical cryoprotectants. In contrast to earlier reports, all the cells studied were found to freeze at temperatures above that indicated for homogeneous nucleation of ice in undercooled liquid water. In the case of human erythrocytes this temperature difference was only 0.5 degrees, but for yeast cells and cells of plant origin the difference amounted to congruent to 9 degrees. Nucleation of ice within the cell (or at the cell wall/membrane) must therefore be initiated by a heterogeneous mechanism. A kinetic analysis of the temperature dependence of nucleation shows the rates to be consistent with the dimensions of the plant cells (or organelles), if these were to be the active nucleators. However, the nucleation kinetics of human erythrocytes are extremely temperature sensitive, and the kinetic parameters only differ by small, though significant, extents from those of the suspension medium. Possible nucleation mechanisms are discussed in terms of the experimental data and the cell dimensions. Finally, one of the underlying assumptions of the kinetic analysis, i.e., that ice growth must be rapid compared to nucleation, has been tested and validated by freeze-fracture electron microscopy.

Calorimetry, Differential Scanning↗