Downscaling cake filtration—Correlations between compressibility of lysozyme crystal filter cakes and filter area

Crystallization is a commonly used method for the purification and formulation of proteins. In order to harmonize crystallization with the mechanical separation process, it is necessary to estimate early the separation conditions of protein crystals. To fulfill this requirement, the feed material for filtration is minimized by reducing the filter area. The filtration behavior of lysozyme crystals in pressure filters with two different filter areas was compared. Crystal slurries with different mean crystal sizes and shapes were produced and the influence of the size and shape on the scalability of filtration data was examined. It was found that for different aggregated crystal structures and isometric crystals, the larger the compressibility of the cake was, the larger were the deviations for the two considered filter areas. For needle‐shaped crystals, the compressibility was not subject to deviations, but the absolute filter cake resistance was. Furthermore, the deviations of the cake resistances increased with higher pressures for all product systems. It was indicated that upscaling from the small filter area is possible for low compressible product systems and at low pressures. With high compressible products and for needle‐like crystals, the filtration time is underestimated with the small filter area.     

Theoretical modeling of filtration of blood cell suspensions

A theoretical model of filtration of suspensions containing red blood cells (RBCs) and white blood cells (WBCs) has been developed. Equations are written for the pressure drop, the filtration flow and the fractions of filter pores containing RBCs (alpha) and WBCs (alpha*). Because the relative resistances (ratios of resistance of cell to resistance of suspending fluid) of RBCs (beta) and WBCs (beta*) through the filter pore are greater than one, the transit of these cells (especially WBCs) through the filter is slower than that of suspending fluid; this leads to values of alpha and alpha* higher than those simply expected from the hematocrit and leukocrit, respectively, in the entering and exiting suspensions. In the absence of pore plugging by the cells (steady flow), the pressure drop can be computed from alpha, alpha*, beta and beta*. In order to model unsteady flow, differential equations are written to include pore plugging and the subsequent unplugging by the rising filtration pressure at a constant flow. By specifying the fractions of entering RBCs (epsilon) and WBCs (epsilon*) which would plug the pores and the rate at which the plugged pores would unplug in response to pressure rise (epsilon u), as well as the fractions of entering RBCs (epsilon p) and WBCs (epsilon p*) that would plug the pores permanently, theoretical pressure-time curves can be generated by numerical integration, and the results fit the experimental data well. From such fitting of theoretical curve to experimental data, information can be deduced for epsilon, epsilon*, epsilon u, epsilon p and epsilon* p.     

Continuous registration of membrane input resistances of small plant cells using a double-pulse current clamp technique for single-electrode impalements : comparison with the conventional two-electrode method

To measure the cell input resistance in Elodea leaf cells, a new single-microelectrode method was explored by comparing the results with conventional two-microelectrode experiments. The new method takes advantage of the difference in the frequency response curves between electrode and cell impedances. By application of electrical stimuli, which contain specific frequency bands, the different impedances can be analyzed separately. To get a distinct separation in the frequency response of cell and electrode, respectively, the electrode capacitance has to be compensated during the impalement. Different time constants of the cell membrane can be accounted for by adjustment of the stimulus length. It is shown that both the single- and the double-electrode method yield the same results, even if the cell input resistances change considerably during the course of the experiment. This demonstrates the usefulness of the new single-electrode method for continuous measurements of cell membrane resistances, especially in cells so small that the double-electrode method is no longer applicable.     

Effect of portacaval surgical anastomosis on systemic and splanchnic hemodynamics in portal hypertensive, cirrhotic rats

The effect of surgical end-to-side portacaval anastomosis (PCSA) on systemic and splanchnic circulation has been studied in cirrhotic rats with portal hypertension (CCl4-phenobarbital method) and in control animals. Hemodynamics have been measured using the microsphere technique, with a reference sample for the systemic hemodynamic measurements, and intrasplenic injection for portal systemic shunting rate measurements. Compared with controls, sham-operated (SO) cirrhotic rats showed a hyperdynamic circulation with increased cardiac output (CO) and decreased mean arterial pressure and peripheral resistances. PCSA in control rats induced only a small change in systemic hemodynamics, with parallel decreases in arterial pressure and peripheral resistances, and a small, nonsignificant increase in CO. In cirrhotic rats, PCSA induced a decrease of CO to values similar to those of control rats, with an increase in total peripheral resistances. PCSA induced an increase in hepatic arterial blood flow in control and in cirrhotic rats, portal pressure becoming in this latter group not different from that of control rats. Blood flow to splanchnic organs was higher in SO cirrhotic than in SO control animals. Thus portal venous inflow was also increased in SO cirrhotic rats. PCSA induced an increase in portal venous inflow in control rats, which was only significant in cirrhotic rats when expressed as a percentage of CO. In SO control animals, a significant correlation was observed between total peripheral resistances and splanchnic arteriolar resistances and between CO and splanchnic blood flow. These correlations were not observed in cirrhotic rats. These results do not support the hypothesis that hyperdynamic circulation shown by cirrhotic rats is based on increases in splanchnic blood flow and (or) massive portal systemic shunting.     

A continuum theory of blood cells filtration at low flow rates

Blood cells filtration with decreasing pressure under gravity was studied for evaluation of the cell fluidity or deformability at a low shear state. A continuum approach was made to the flow and pressure in the filter at the low flow state to relate macro- and micro-scopic quantities. The mass conservation law of each species provided a set of differential equations with respect to the pore fraction and filter resistance. The numerical calculation was made for various values of hematocrit and leukocrit. It was shown that the filter resistance might be increased with decreasing pressure, resulting from both red and white cells. The leukocrit, more than 0.05% white cells, may influence the filtration, depending upon the cell deformation. Even in the absence of the white cell, a decrease in pressure increased the filter resistance markedly. The present result indicates that single red cell shows a nonlinear behavior of flow in pores at the low pressure level.     

Impact of Low Filter Resistances on Subjective and Physiological Responses to Filtering Facepiece Respirators

Ten subjects underwent treadmill exercise at 5.6 km/h over one hour while wearing each of three identical appearing, cup-shaped, prototype filtering facepiece respirators that differed only in their filter resistances (3 mm, 6 mm, and 9 mm H₂O pressure drop). There were no statistically significant differences between filtering facepiece respirators with respect to impact on physiological parameters (i.e., heart rate, respiratory rate, oxygen saturation, transcutaneous carbon dioxide levels, tympanic membrane temperature), pulmonary function variables (i.e., tidal volume, respiratory rate, volume of carbon dioxide production, oxygen consumption, or ventilation), and subjective ratings (i.e., exertion, thermal comfort, inspiratory effort, expiratory effort and overall breathing comfort). The nominal filter resistances of the prototype filtering facepiece respirators correspond to airflow resistances ranging from 2.1 - 6.6 mm H₂O/L/s which are less than, or minimally equivalent to, previously reported values for the normal threshold for detection of inspiratory breathing resistance (6 - 7.6 mm H₂O/L/sec). Therefore, filtering facepiece respirators with filter resistances at, or below, this level may not impact the wearer differently physiologically or subjectively from those with filter resistances only slightly above this threshold at low-moderate work rates over one hour.     

Surprisingly fast disappearance of beta-lactam selection pressure in cultivation as detected with novel biosensing approaches

Tetracycline and beta-lactam resistances among others are used as selection markers in the production of recombinant proteins. The beta-lactam resistance is based on degradation, i.e. the selection pressure gradually disappears from the culture, whereas tetracycline resistance is based on active efflux. We have studied the kinetics of the stability of antibiotic selection pressure in culture using a simple model system (pBR322 in Escherichia coli). Concentrations of ampicillin, carbenicillin and tetracycline were measured with novel sensor cells developed in our lab. These cells are specifically induced to produce light in the presence of the drugs and here their performance was shown to be excellent in monitoring antibiotic concentrations in cell culture. The sensor cells are cheap to produce and use and a high number of samples can be analysed simultaneously. To our surprise, ampicillin and carbenicillin were completely degraded after 2.5-3.0 h of culture, although it has been widely claimed that especially carbenicillin is a good selective agent, whereas tetracycline was stable in culture. beta-lactamase activity in culture was found to correlate with the kinetics of ampicillin degradation.     

Flow characteristics of red cell containing fluids through pores. The effect of filter plugging, a mathematical model

A mathematical model was developed that describes the effects of filter plugging on flow through 3 micron pore polycarbonate filters as a function of time, pressure, and cell concentration, both under stirring and nonstirring conditions. The mathematical constants for the model were derived from experimental data generated with a filtration apparatus, and were tested by using various concentrations of cells that are able to plug filter pores. A computer simulation program was written to test the model over a wide range of nonfilterable cell concentrations.     

Radial hydraulic conductivity along developing onion roots

Although most studies have shown that water uptake varies along the length of a developing root, there is no consistent correlation of this pattern with root anatomy. In the present study, water movement into three zones of onion roots was measured by a series of mini-potometers. Uptake was least in the youngest zone (mean hydraulic conductivity, Lpr = 1.5 x 10(-7) +/- 0.34 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots) in which the endodermis had developed only Casparian bands and the exodermis was immature. Uptake was significantly greater in the middle zone (Lpr = 2.4 x 10(-7) +/- 0.43 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots) which had a mature exodermis with both Casparian bands and suberin lamellae, and continued at this level in the oldest zone in which the endodermis had also developed suberin lamellae (Lpr = 2.8 x 10(-7) +/- 0.30 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots). Measurements of the hydraulic conductivities of individual cells (Lp) in the outer cortex using a cell pressure probe indicated that this parameter was uniform in all three zones tested (Lp = 1.3 x 10(-6) +/- 0.01 x 10(-6) m MPa-1 s-1; +/- SE, n = 60 cells). Lp of the youngest zone was lowered by mercuric chloride treatment, indicating the involvement of mercury-sensitive water channels (aquaporins). Water flow in the older two root zones measured by mini-potometers was also inhibited by mercuric chloride, despite the demonstrated impermeability of their exodermal layers to this substance. Thus, water channels in the epidermis and/or exodermis of the older regions were especially significant for water flow. The results of this and previous studies are discussed in terms of two models. The first, which describes maize root with an immature exodermis, is the 'uniform resistance model' where hydraulic resistances are evenly distributed across the root cylinder. The second, which describes the onion root with a mature exodermis, is the 'non-uniform resistance model' where resistances can be variable and are concentrated in a certain layer(s) on the radial path.     

Telemetered renal responses in dogs during detection of explosives.

The primary objectove of this work was to develop and test a method of measuring and characterizing renal hemodynamic responses in unrestrained dogs. Recordings of left kidney blood flow and abdominal aortic pressure were obtained from unrestrained dogs through the use of a two-channel implanted telemetry system during episodes of search and detection of simulated explosives. In each of a number of sequences, the dog was first given a start signal, and after locating the hidden device, was rewarded with food. Data were assessed at the start, find, and recovery segments. The dynamic flow and pressure, together with a hydraulic renal model, were used to derive the total preglomerular and postglomerular vascular resistances and the mean level of glomerular hydrostatic pressure. Data from three dogs obtained by telemetry and analyzed through the use of the model have shown that compared to the start of the test, the location of hidden explosives results in a decrease in both the level of mean aortic blood pressure and the preglomerular resistance; whereas, the reward results in an elevation of mean blood pressure and preglomerular resistance. The postglomerular resistance varied less than the preglomerular resistance, and mean flow did not vary significantly. This work has shown that the stimulation of a reward and the successful performance of a task lead to significant renal responses in dogs. It has further shown that telemetry, when employed with improved data analysis techniques, permits renal hemodynamics to be assessed in unrestrained animal subjects.     

The mechanism of beta-adrenergic receptor blockade-induced elevation of arterial blood pressure in rats

Beta-adrenergic blockade increases peripheral vascular resistances acutely and paradoxically lowers arterial blood pressure in most species but not in the rat. In this study the hypothesis has been tested of a significant participation of unopposed alpha-adrenergic mediated vasoconstriction following beta-adrenergic blockade in blood pressure regulation of conscious rats. Alpha-adrenergic blockade in propranolol-pretreated rats significantly decreased mean arterial blood pressure by 22%, heart rate by 20%, and total peripheral resistance by 14% when compared to rats treated only with propranolol, whereas cardiac output was similar in the two groups. A significant 28% reduction of coronary blood flow in rats treated with alpha- and beta-adrenergic blockers, when compared to rats treated only with propranolol, is most likely related to phentolamine's indirect effects on the coronary vasculature. Cerebrovascular and renovascular resistances were similar in these groups. All changes reported were significant at the 95% confidence level. It is concluded that the mechanism of increased blood pressure following beta-adrenergic blockade in rats is related at least in part to unmasking of unopposed alpha-receptor tone, which, however, is minimal in the coronary, cerebral and renal circulations.     

Transepithelial resistance of ciliary epithelial cells in culture: functional modification by protamine and extracellular calcium.

1. Bovine pigmented and human non-pigmented ciliary epithelial cells were cultured on porous filter supports to obtain measurements of transepithelial electrical parameters. 2. The non-pigmented cells showed maximal transepithelial resistance of 15-30 omega cm2 from the third to seventh day in culture. 3. The pigmented ciliary cells reached maximal resistances of 9-20 omega cm2 after the fourth day in culture. 4. The transepithelial resistances of the cultured epithelia were functionally increased by protamine. This effect could be reversed by heparin. 5. We conclude that the range of resistances in cultured ciliary epithelial cells is the same as in whole ciliary preparations. Thus, cultured ciliary epithelial cells can be used for studies on transepithelial transport.     

Determination of erythrocyte transit times through micropores. I--Basic operational principles

To study the transit times of each red blood cell passing through cylindrical micropores and in order to evaluate sub-population of cells with regard to their deformability, we have developed a new system called the cell transit time analyser (CTTA). By using an AC voltage (100 KHz) across a special filter, we measure the electrical conductance change produced by the cells passing through the pores under a known driving pressure. This computer based device provides the distribution of transit times tau for 2000 cells in 1 minute and as a result the mean transit time [tau]. Experiments with red cells were designed to evaluate the flow behavior of both normal cells and cells whose mechanical properties were artificially altered. Cell volume was changed by use of non-isotonic media. Cell shape and cell volume were modified by varying the pH of the suspending buffer. Results of these experiments are: 1) a skew distribution of transit times towards high tau values for both control cells and artificially altered cells is observed: 2) [tau] is minimum for isotonic conditions and increases sharply for either hypotonic or hypertonic media: 3) [tau] is minimum at physiological pH and increases for either acid or alcaline changes of pH.     

Genesis of the monophasic action potential: role of interstitial resistance and boundary gradients

The extracellular potential at the site of a mechanical deformation has been shown to resemble the underlying transmembrane action potential, providing a minimally invasive way to access membrane dynamics. The biophysical factors underlying the genesis of this signal, however, are still poorly understood. With the use of data from a recent experimental study in a murine heart, a three-dimensional anisotropic bidomain model of the mouse ventricular free wall was developed to study the currents and potentials resulting from the application of a point mechanical load on cardiac tissue. The applied pressure is assumed to open nonspecific pressure-sensitive channels depolarizing the membrane, leading to monophasic currents at the electrode edge that give rise to the monophasic action potential (MAP). The results show that the magnitude and the time course of the MAP are reproduced only for certain combinations of local or global intracellular and interstitial resistances that form a resting tissue length constant that, if applied over the entire domain, is smaller than that required to match the wave speed. The results suggest that the application of pressure not only causes local depolarization but also changes local tissue properties, both of which appear to play a critical role in the genesis of the MAP.     

The water relations of the root–soil interface

The difference in hydrostatic pressure between the xylem of the leaf and the soil depends, for a given transpiration rate, on the series of hydraulic resistances encountered along this pathway. Many studies have shown that the sum of the resistances in the plant and the soil is too small to account for the fall in water pressure between the leaf xylem and the soil, especially when plants are growing in sandy soils, which are prone to dry rapidly. A resistance at the root–soil interface, caused possibly by poor contact between the roots and the soil, has been proposed to account for the discrepancy. We explored the resistance in the pathway from soil to leaf using a technique that allows precise and continuous non-destructive measurement of the hydrostatic pressure in the leaf xylem. When the soil was leached with water, the fall in leaf water status as the soil dried was reasonably well described by a simple physical model without the need to invoke an interfacial resistance. However, when the soil was flushed with a nutrient solution with an osmotic pressure of 70kPa, the hydrostatic pressure in the leaf xylem fell several times faster than that in the soil. We suggest that solutes accumulated either in the root or just outside it, creating large osmotic pressures, which gave the appearance of an interfacial resistance.     

Hemodynamic and renal effects of acute and progressive nitric oxide synthesis inhibition in anesthetized dogs

This study evaluated the effects of progressive nitric oxide (NO) inhibition in the regulation of systemic and regional hemodynamics and renal function in anesthetized dogs. The N(G)-nitro-L-arginine methyl ester group (n = 9) received progressive doses of 0.1, 1, 10, and 50 microg. kg(-1). min(-1). Renal (RBF), mesenteric (MBF), iliac (IBF) blood flows, mean arterial pressure (MAP), pulmonary pressures, cardiac output (CO), and systemic and pulmonary vascular resistances were measured. During N(G)-nitro-L-arginine methyl ester infusion, MAP and systemic vascular resistances increased in a dose-dependent manner. Mean pulmonary pressure and pulmonary vascular resistances increased in both the N(G)-nitro-L-arginine methyl ester and the control group, but the increase was more marked in the N(G)-nitro-L-arginine methyl ester group during the last two infusion periods. CO decreased progressively, before any significant change in blood pressure was noticeable in the N(G)-nitro-L-arginine methyl ester group. IBF decreased significantly from the first N(G)-nitro-L-arginine methyl ester dose, whereas RBF and MBF only decreased significantly during the highest N(G)-nitro-L-arginine methyl ester dose. Urinary volume and sodium excretion only increased significantly in the time control group during the two last time periods. The pulmonary vasculature was more sensitive than the systemic vasculature, whereas skeletal muscle and renal vasculatures showed a greater sensitivity to the inhibition of NO production than the mesenteric vasculature. NO synthesis inhibition induces a progressive antidiuretic and antinatriuretic effect, which is partially offset by the increase in blood pressure.     

Theoretical analysis of the noncardiac limits to maximum exercise

When right atrial pressure (Pra) is greater than zero (atmospheric pressure), cardiac output is determined by the intersection of two functions, cardiac function and return function, which is used here to mean the determinants of venous return. When Pra < or = 0, flow is only determined by circuit function. The objective of this analysis was to determine the potential changes in return function that need to occur to allow the maximum cardiac output during exercise when Pra < or = 0 or is constant. The analysis expands on the model of Green and Jackman and includes the effects of changes in circuit parameters, including venous resistance, changes in capacitance, and muscle contractions. The analysis is based on the model of the circulation proposed by Permutt and co-workers, which assumes that the systemic circulation has two lumped compliant regions in parallel with independent inflow and outflow resistances. Changes in total flow in this model can come about by changes in the distribution of flow between the regions, recruitment of unstressed vascular volume, and changes in the regional venous resistances. The data for the analysis are from previous animal studies and are normalized to a 70-kg man. The major conclusions are that, to achieve the high cardiac output that occurs at peak exercise, there need to be marked changes in the distribution of blood flow, recruitment of unstressed volume, and the venous resistance draining vascular beds. A consequence of the increase in peripheral flow is a marked increase in pressure in the veins of the working muscle. Muscle contractions are potentially a very important mechanism for transiently decreasing this pressure and preventing excessive filtration of plasma during exercise.     

Localization of the sites of pulmonary vasomotion by use of arterial and venous occlusion

In this study, we present a new approach for using the pressure vs. time data obtained after various vascular occlusion maneuvers in pump-perfused lungs to gain insight into the longitudinal distribution of vascular resistance with respect to vascular compliance. Occlusion data were obtained from isolated dog lung lobes under normal control conditions, during hypoxia, and during histamine or serotonin infusion. The data used in the analysis include the slope of the arterial pressure curve and the zero time intercept of the extrapolated venous pressure curve after venous occlusion, the equilibrium pressure after simultaneous occlusion of both the arterial inflow and venous outflow, and the area bounded by equilibrium pressure and the arterial pressure curve after arterial occlusion. We analyzed these data by use of a compartmental model in which the vascular bed is represented by three parallel compliances separated by two series resistances, and each of the three compliances and the two resistances can be identified. To interpret the model parameters, we view the large arteries and veins as mainly compliance vessels and the small arteries and veins as mainly resistance vessels. The capillary bed is viewed as having a high compliance, and any capillary resistance is included in the two series resistances. With this view in mind, the results are consistent with the major response to serotonin infusion being constriction of large and small arteries (a decrease in arterial compliance and an increase in arterial resistance), the major response to histamine infusion being constriction of small and large veins (an increase in venous resistance and a decrease in venous compliance), and the major response to hypoxia being constriction of the small arteries (an increase in arterial resistance). The results suggest that this approach may have utility for evaluation of the sites of action of pulmonary vasomotor stimuli.     

Diffusion and the limiting substrate in two-substrate immobilized enzyme systems

The effects of mass transport resistances on two-substrate immobilized enzyme systems are investigated theoretically. It is shown that the effects of mass transport resistances on the overall reaction rate are related mainly to the transport of the limiting substrate. In the absence of external mass transport resistances, the limiting substrate can be identified by knowing only the ratio of the bulk substrate concentrations, the permeability of the support to the two substrates, and the stoichiometry of the reaction. However, a combination of internal and external mass transport resistances may result in the other substrate becoming limiting. These effects are most significant when the mass transport resistances are high. Applications in the design of enzyme electrodes and chemical reactors are discussed.     

Comparison of vascular and respiratory effects of endothelin-1 in the pig

The haemodynamic and respiratory responses caused by i.v. administration of endothelin-1 (ET-1) (20-100 pmol/kg) were studied in anaesthetized spontaneously breathing pigs. Intravenous bolus administration of synthetic ET-1 (40-100 pmol/kg) caused a transient decrease followed by a long-lasting increase in mean pulmonary arterial pressure and dose dependent vasoconstriction both in the systemic and pulmonary circulations. The effect on pulmonary arterial pressure was biphasic, with an initial transient fall followed by a long-lasting dose dependent increase. A biphasic response of the systemic mean arterial pressure was demonstrated only at a high dose of ET-1 (100 pmol/kg). ET-1 administration did not significantly change breathing pattern or phasic vagal input, but caused a significant decrease in passive compliance. Passive resistances or active compliance and resistances of the respiratory system were not modified. These results suggest that in the pig ET-1 is a more potent constrictor of vascular than of bronchial smooth muscle. The vasoconstrictor activity was greater in the pulmonary than the systemic circulations.