Quantitative Examination of Tissue Concentration Profiles Associated with Microdialysis

Spatial solute concentration profiles resulting from in vivo microdialysis were measured in rat caudate-putamen by quantitative autoradiography. Radiolabeled sucrose was included in the dialysate, and the tissue concentration profile measured after infusions of 14 min and 61.5 min in an acute preparation. In addition, the changes in sucrose extraction fraction over time were followed in vivo and in a simple in vitro system consisting of 0.5% agarose. These experimental results were then compared with mathematical simulations of microdialysis in vitro and in vivo. Simulations of in vitro microdialysis agreed well with experimental results. In vivo, the autoradiograms of the tissue concentration profiles showed clear evidence of substantial differences between 14 and 61.5 min, even though the change in extraction fraction was relatively small over that period. Comparison with simulated results showed that the model substantially underpredicted the observed extraction fraction and overall amount of sucrose in the tissue. A sensitivity analysis of the various model parameters suggested a tissue extracellular volume fraction of approximately 40% following probe implantation. We conclude that the injury from probe insertion initially causes disruption of the blood-brain barrier in the vicinity of the probe, and this disruption leads to an influx of water and plasma constituents, causing a vasogenic edema.     

Modelling Thrombin Generation in Human Ovarian Follicular Fluid

A mathematical model is constructed to study thrombin production in human ovarian follicular fluid. The model results show that the amount of thrombin that can be produced in ovarian follicular fluid is much lower than that in blood plasma, failing to reach the level required for fibrin formation, and thereby supporting the hypothesis that in follicular fluid thrombin functions to initiate cellular activities via intracellular signalling receptors. It is also concluded that the absence of the amplification pathway to thrombin production in follicular fluid is a major factor in restricting the amount of thrombin that can be produced. Titration of the initial concentrations of the various reactants in the model lead to predictions for the amount of tissue factor and phospholipid that is required to maintain thrombin production in the follicle, as well as to the conclusion that tissue factor pathway inhibitor has little effect on the time that thrombin generation is sustained. Numerical experiments to determine the effect of factor V, which is at a much reduced level in follicular fluid compared to plasma, and thrombomodulin, illustrate the importance for further experimental work to determine values for several parameters that have yet to be reported in the literature.     

Competition in a pHauxostat

Summary Competition between a rapidly growing bacterium and a slower growing yeast was studied in a bioreactor with feed rate controlled by the rate of consumption of substrate. The dilution rate increase indicates the take over by the faster organism. Simple models track well with the actual data.     

Activation of transglutaminase at calcium levels consistent with a role for this enzyme as a calcium receptor protein

The sensitivity of tissue transglutaminase to activation by Ca²⁺ and other cellular factors was investigated using the enzyme purified from rat liver. The inclusion of Mg²⁺ in the assay system appeared to reduce the Ca²⁺-requirement of the enzyme when native N,N-dimethylcasein was used as the protein acceptor substrate. However, when this protein was dephosphorylated, the Ca²⁺-requirement was unaffected by Mg²⁺. In addition, using this modified assay, a K_m for Ca²⁺ was calculated to be in the range of 3–4 M, at least an order of magnitude lower than that obtained with native acceptor substrate. Membrane phospholipids, 1,2-diolein and calmodufin were found not to affect the activation oftransglutaminase by Ca²⁺. The sensitivity of transglutaminase to Ca²⁺ which we have now demonstrated suggests that this enzyme may directly act as a receptor protein for Ca²⁺ during stimulusresponse coupling mediated by this cation.     

Hanging Magnetic Stirrer Which Minimizes Cell Disruption

A magnetic stirring bar enclosed in tubing forms a large paddle which produces good mixing at slow rotational speeds. It is suspended above the bottom of the vessel and cannot grind cells or become misaligned.     

Primary antibody-forming cells and secondary B cells are generated from separate precursor cell subpopulations

Two precursor cell subpopulations have been isolated from the spleen cells of nonimmune mice. The major B cell subpopulation binds high levels of the J11D monoclonal antibody and, upon T cell-dependent antigenic stimulation, gives rise to primary antibody-forming cell clones but not secondary B cells. A minority of the 10%-14% of Ia+ precursors that bind low levels of J11D (J11Dlo) also generate antibody-forming cell clones after primary stimulation. However, over 70% of J11Dlo precursors yield no primary antibody-forming cell clones but instead give rise to secondarily responsive B cells. The existence of a distinct precursor cell subpopulation that is responsible for the generation of B cell memory is further evidenced by the distribution of variable region clonotypes among J11Dlo primary precursors, which resembles the clonotype patterns of secondary B cells, and by the accumulation of somatic mutations in their clonal progeny.