Rat Hippocampal Lactate Efflux During Electroconvulsive Shock or Stress Is Differently Dependent on Entorhinal Cortex and Adrenal Integrity

The role of the entorhinal cortex and the adrenal gland in rat hippocampal lactate formation was assessed during and after a short-lasting immobilization stress and electroconvulsive shock (ECS). Extracellular lactate was measured on-line using microdialysis and enzyme reactions (a technique named lactography); in some rats, unilateral lesions of the entorhinal cortex were made or the bilateral adrenal glands were removed. The stress-evoked increase in hippocampus lactate was not altered either ipsi- or contralateral to an entorhinal cortex lesion. The response to ECS was attenuated only in the hippocampus ipsilateral to the entorhinal cortex lesion. Removal of bilateral adrenal glands caused some delay in the increase in hippocampal lactate after ECS and a major reduction in the stress-evoked lactate response. These results indicate that (1) the entorhinal cortex is activated by ECS, thereby activating hippocampal lactate efflux and presumably metabolism, and (2) the adrenal gland is essential in the response to stress and, to a minor extent, in the ECS-altered hippocampal metabolism.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

Effect of sex hormones on human T cell activation by concanavalin A

The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.     

Immobilized enzyme cellulose hollow fibers: I. Immobilization of heparinase

The immobilization of heparinase to tresyl-chloride-activated cellulose hollow fibers for the removal of heparin from the bloodstream was examined. Whole blood can be circulated through cellulose hollow fibers without hemolysis and the tresyl chloride chemistry provides a strong linkage which limits the release of the enzyme from the support. The tresylation and immobilization methods were modified and optimized to improve the heparinase activity retained by cellulose. Pretreatment of the hollow fibers with 0.05/V sodium hydroxide increased the degree of tresylation and the immobilization yield by a factor of five. The use of triethylamine as the organic base in the tresyl chloride activation resulted in threefold greater activity retention by the support than when pyridine was used. Together, sodium hydroxide pretreatment and triethylamine enhanced the activity retained by cellulose to 26.2 +/- 7.0% of that bound to the support. The activity retention was also a function of the technique used for immobilization. The best results were achieved when the enzyme was applied to the activated fibers once every 12 to 24 h for a total of four times. The active enzyme loading on the fibers was 0.3 mg heparin degraded/h cm(2) when 4.5 mug protein/cm(2) was bound to the fibers.     

Transport rates of proteins in porous materials with known microgeometry.

Many biological and biotechnological systems involve the diffusion of macromolecules through complicated macroporous (pore size on the order of 10-100 microns) environments. In this report, we present and evaluate an experimental system for measuring the rate of protein transport in an inert, macroporous membrane. For this particular membrane system, the microgeometry was characterized in terms of distribution of pore size, position, and orientation. Although the rate of protein desorption was much less than expected based on continuum diffusion models, we demonstrate that the measured transport rates are consistent with diffusion of protein in a complex, interconnected network of water-filled pores. The porous systems exhibit transitional behavior in quantitative agreement with the behavior of percolation lattices (mean square error 7%, n = 29). Predictive mathematical models of the diffusion process were developed: these models used percolation concepts to describe pore topology, continuum models of diffusion/dissolution to describe protein movement at each single pore, and measured pore size distributions. Effective diffusion coefficients for protein transport in aqueous, constricted macropores were predicted by this technique. Predicted diffusion coefficients, based on measured and derived microstructural parameters, agree with experimentally measured diffusion coefficients within a factor of 2. This approach may be useful in the design of porous polymer systems for biological applications and for evaluating other biological systems where conduction of mass, heat, momentum, or charge occurs in a heterogeneous environment.     

Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis

Immobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic K(m) value for the system. The intrinsic K(m) value for heparinase-cellulose is 0.4 +/- 0.3 mg/mL (N = 6) and it is the same order of magnitude as the K(m) value for soluble heparinase.     

Calcium concentration and movement in the ventricular cardiac cell during an excitation-contraction cycle.

This paper extends the model for Ca movement in the cardiac ventricular cell from the diadic cleft space to the entire sarcomere. The model predicts the following: 1) Shortly after SR release there is a [Ca] gradient >3 orders of magnitude from cleft center to M-line which, 50 ms after release, is still >30. Outside the cleft, 40 ms after cessation of release, the axial gradient from Z to M-line is >3. 2) At the end of SR release, >50% of the total Ca released is bound to low-affinity inner sarcolemmal phospholipid binding sites within the cleft. 3) Halving the SR release almost doubles the fraction of release removed from the cell via Na/Ca exchange and reduces average sarcomeric free [Ca] by 70%. 4) Adding 100 microM fluo-3, which doubles the buffering capacity of the cytoplasm, reduces peak average sarcomeric [Ca] by >50% and increases the initial half-time for [Ca] decrease by approximately twofold. 5) A typical Ca "spark" can be generated by an SR release 20% of maximum (4 x 10(-20) moles) over 2 ms. Fluo-3 (100 microM) significantly "shrinks" the spark. 6) The "spark" is a consequence of elementary events within the diadic cleft space. For example, removal of cleft binding sites would cause average sarcomeric Ca to increase by >10 fold, fall 10 times more rapidly, decrease latency for appearance of the spark by >20 times, and reduce spark duration by 85%. 7) Dividing SR Ca release between cleft and corbular SR produces a secondary [Ca] peak and a "flattening" of the sarcomeric [Ca] transient. These changes probably could not be resolved with current confocal microscopic techniques.     

A novel biotinylated degradable polymer for cell-interactive applications

We previously demonstrated that the lambda system integrated into the host chromosome can overcome the instability encountered in continuous operations of unstable plasmid-based expression vectors. High stability of a cloned gene in a lysogenic state and a high copy number in a lytic state provide cloned-gene stability and overexpression in a two-stage continuous operation. But the expression by the commonly used S- mutant lambda was only twice as high as that of the single copy. To increase the expression in the lambda system, we constructed a Q- mutant lambda vector that can be used in long-term operations such as a two-stage continuous operation. The Q- mutant phage lambda is deficient in the synthesis of proteins involved in cell lysis and lambda DNA packaging, while the S- mutant is deficient in the synthesis of one of two phage proteins required for lysis of the host cell and liberation of the progeny phage. Therefore, it is expected that the replicated Q- lambda DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of the cloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products. The beta-galactosidase expression per unit cell by the Q- mutant in a lytic state was about 30 times higher than that in a lysogenic state, while the expression by the commonly used S- mutant in a lytic state was twice as high as that in a lysogenic state. The optimal switching time of the Q- mutant from the lysogenic state to the lytic state for the maximum production of beta-galactosidase was 5.3 h, which corresponds to an early log phase in the batch operation.     

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca²⁺-dependent, but not completely reversible, as on removal of Ca²⁺ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca²⁺ in a micromolar range, with half maximum activation at about 3×10⁻⁶ M. The protein becomes inactivated when Ca²⁺ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2–4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.     

Investigation of a whole blood fluidized bed Taylor-Couette flow device for enzymatic heparin neutralization

The use of clinical bioreactors will increase as more therapeutic proteins are being cloned, expressed, and produced at a reduced cost. The proposed use of an immobilized heparinase I reactor to make heparin anticoagulation a safer therapy is an example of how the specificity and high activity of an enzyme could be incorporated into a system to ultimately benefit a patient. However, the development of a safe and efficient bioreactor is important for the use of immobilized heparinase I and other therapeutic proteins designed for use in medical extracorporeal procedures. This study examined the possibility of using Taylor-Couette flow and "flow-induced" recirculation of the agarose beads as a way to fluidize agarose-bound heparinase in whole blood. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. A reactor based on Taylor-Couette flow was designed and modified with a tangential recirculation line. The reactor was tested for efficacy and safety in vitro in human blood. Visualization studies in water and 42% glycerol were used to determine the minimum rotation rate for efficient fluidization. The strategic placement of the recirculation line allowed recirculation of the agarose without the use of an external pump. The device removed 90% of the heparin activity within 2 min from 450 cc of human blood at a blood flow rate of 100 mL/min. Furthermore, the device maintained inlet and outlet clotting times of 269 +/- 10 and 235 +/- 6 s, respectively, demonstrating the potential for regional heparinization. Blood damage was a function of gel volume fraction and rotation rate of the inner cylinder. Hemolysis of the red cells is an important issue when Taylor vortices are combined with macroscopic solid particles such as agarose beads. A modified Taylor-Couette flow device was developed to treat whole blood and operational criteria were established to minimize hemolysis.