A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. III. Influence of the matrix composition on the lead phosphate precipitation process in acid phosphatase cytochemistry

Synopsis A model system for the study of the dynamics of precipitation processes, described elsewhere and consisting of polyacrylamide films, was used to investigate the influence of the composition of the matrix, in which precipitation occurs, on the lead phosphate precipitation process in acid phosphatase cytochemistry. The situation at an enzymatic site can be simulated by pumping a phosphate-containing solution and the Gomori medium for acid phosphatase (lead containing medium) along opposite sides of a polyacrylamide film. The procipitation of lead phosphate was found to start at a lower phosphate concentration of the solution flowing along films into which histone, casein, RNA or polygalacturonate had been incorporated, than in control films. DNA, chondroitin sulphate and bovine serum albumin (unfixed) did not give this effect. Fixed bovine serum albumin incorporated into the film slightly decreased the phosphate concentration at which a precipitate appeared. Nuclear staining occurring under suboptimal conditions for phosphate trapping is probably due to a local matrix effect. The model studies suggested DNA-associated phosphorylated histones and phosphoproteins as likely candidates for such an effect, and RNA as a less likely one. Artefactual precipitates at the plasma membrane might be due to carboxyl groups of sialic acid.     

A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. II. Effect of the composition of the incubation medium on the trapping of phosphate ions in acid phosphatase cytochemistry

Synopsis A model system developed for the study of the dynamics of capture reactions for diffusable compounds in cytochemistry served as a basis for the experiments reported in the present paper. The model was used to study the effect of the composition of the cytochemical medium on the trapping of phosphate ions by lead (II) ions in acid phosphatase cytochemistry. In this system a phosphate-containing solution and a lead-containing solution (cytochemical medium) are pumped along opposite sides of a polyacrylamide film. The phosphate concentration at which measurable precipitation starts in the film (critical phosphate concentration) was taken as a measure of the trapping efficiency of the cytochemical medium. The addition of -glycerophosphate and cytidine-5-monophosphate to a buffered lead-containing solution resulted in a higher critical phosphate concentration. Both substrates had an effect on the crystal form of lead phosphate. The addition of chloride ions and acetone, as well as decreasing the molarity of the acetate buffer of the cytochemical medium, were found to lower the critical phosphate concentration, whereas the addition of fluoride ions, glucose, and sucrose had no effect. From the effect of variations in the composition of the cytochemical medium on the trapping efficiency and the turnover number of acid phosphatase in the medium, it was possible to predict which cytochemical medium would be the most suitable for the demonstration of acid phosphatase activity in guinea-pig peritoneal exudate cells. The results were in accordance with the localization of acid phosphatase activity: the higher the trapping efficiency and the turnover number, the higher the amount of precipitate and the number of positive enzymatic sites. In this way an improved cytochemical medium for acid phosphatase was developed.     

Enzyme layers on glass as a new model for the quantitative study of capture reactions in cytochemistry,with special attention to acid phosphatase

Summary A model system is described for the study of capture reactions for diffusable compounds in enzyme cytochemistry. The model, which allows the investigation of the influence of the composition of the cytochemical medium, the enzymatic activity, and the dimensions of the enzymatic site on the capture reaction, consists of very thin homogeneous layers of enzyme (0.01–0.1 m thick) on glass, which are incubated in the cytochemical medium. The fraction of the total amount of liberated product precipitated in the enzyme layer is dependent not only on the trapping efficiency of the cytochemical medium but also on the concentration of the primary reaction product that can be built up in the enzyme layer. Calculations were performed to determine the steady-state concentration of the primary reaction product that can be built up in the enzyme layer. Acid phosphatase was used as enzyme. The problems associated with the model and its applicability to other types of cytochemical reactions are discussed.     

A mathematical model of the Calvin photosynthesis cycle

1. A mathematical model is presented for photosynthetic carbohydrate formation in C3 plants under conditions of light and carbon dioxide saturation. The model considers reactions of the Calvin cycle with triose phosphate export and starch production as main output processes, and treats concentrations of NADPH, NAD+, CO2, and H+ as fixed parameters of the system. Using equilibrium approximations for all reaction steps close to equilibrium steady-state and transient-state relationships are derived which may be used for calculation of reaction fluxes and concentrations of the 13 carbohydrate cycle intermediates, glucose 6-phosphate, glucose 1-phosphate, ATP, ADP, and inorganic (ortho)phosphate. 2. Predictions of the model were examined with the assumption that photosynthate export from the chloroplast occurs to a medium containing orthophosphate as the only exchangeable metabolite. The results indicate that the Calvin cycle may operate in a single dynamically stable steady state when the external concentration of orthophosphate does not exceed 1.9 mM. At higher concentrations of the external metabolite, the reaction system exhibits overload breakdown; the excessive rate of photosynthate export deprives the system of cycle intermediates such that the cycle activity progressively approaches zero. 3. Reactant concentrations calculated for the stable steady state that may obtain are in satisfactory agreement with those observed experimentally, and the model accounts with surprising accuracy for experimentally observed effects of external orthophosphate on the steady-state cycle activity and rate of starch production. 4. Control analyses are reported which show that most of the non-equilibrium enzymes in the system have a strong regulatory influence on the steady-state level of all of the cycle intermediates. Substrate concentration control coefficients for cycle enzymes may be positive, such that an increase in activity of an enzyme may raise the steady-state concentration of the substrate is consumes. 5. Under optimal external conditions (0.15-0.5 mM orthophosphate), reaction flux in the Calvin cycle is controlled mainly by ATP synthetase and sedoheptulose bisphosphatase; the cycle activity approaches the maximum velocity that can be supported by the latter enzyme. At lower concentrations of external orthophosphate the cycle activity is controlled almost exclusively by the phosphate translocator.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.     

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity.

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.     

The Hazard of Acid Differentiation in Gomori's Method for Acid Phosphatase

In his original method for the histochemical demonstration of acid phosphatase, Gomori prescribed differentiation of incubated sections by rinsing them in 14% aqueous acetic acid, to remove the nonspecifically precipitated lead deposits. According to him, the enzymatically produced lead phosphate is not washed out by this procedure. As a result of recent improvements in tissue preparation and shorter incubation time, this staining reaction as it is used now is quite sensitive to an acetic acid wash. If this wash is used as recommended originally, it may completely abolish a truly positive reaction. To avoid falsely negative results, and to compare sections of normal and pathological tissue, omission of this differentiation by acetic acid is essential. The risk of mistaking nonspecific lead precipitates in the interpretation of a positive reaction is very small, and can be avoided by running a negative control slide in which no lead phosphate can be produced enzymatically.     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

Studies on the phenazine methosulphate-tetrazolium salt capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. I. Localization artefacts caused by the escape of reduced co-enzyme during cytochemical reactions for NAD(P)+-dependent dehydrogenases

The correct localization of oxidative enzymes using cytochemical tetrazolium methods, in which low molecular weight electron carriers such as NAD(P)H and reduced phenazine methosulphate (PMSH) are used, can be endangered by the escape of the reduced intermediates before they react to form the insoluble formazan at the true enzyme-containing sites. To investigate this phenomenon, the glucose-6-phosphate dehydrogenase reaction was studied in fixed erythrocytes which, because of their microscopic dimensions, are well-suited for studying the loss of intermediates. A mixture of active and heat-inactivated fixed erythrocytes was incubated in a PMS-supplemented medium for glucose-6-phosphate dehydrogenase. The cytophotometric histograms showed that the final formazan precipitate was equally distributed over both active and inactivated cells. When bovine serum albumin was added to the medium, all the formazan was found to be bound to this protein and the erythrocytes remained essentially unstained. The false localization in this system could be explained by an unfavourable balance between the capture of electrons carried by NADPH within the erythrocyte and the diffusion of NADPH out of the erythrocyte. The rate constant of NADPH oxidation was determined, as was also the diffusion constant of NADPH in a protein matrix. Substituting the data obtained into formulae derived from the enzyme cytochemical localization theory of Holt & O'Sullivan (1958), it was calculated that the capture reaction was highly deficient and, theoretically, less than 1% of the total amount of formazan produced was localized within the erythrocyte which explains the false localization observed. The importance of these findings for the cytochemical demonstration of NAD(P)+-dependent dehydrogenases in cells and electropherograms is briefly discussed.     

Regulatory kinetics of wheat-germ aspartate transcarbamoylase. Adaptation of the concerted model to account for complex kinetic effects of uridine 5''-monophosphate.

The kinetic effects of the end-product inhibitor UMP on aspartate transcarbamoylase (EC 2.1.3.2) purified to homogeneity from wheat germ were studied. In agreement with an earlier study of the relatively crude enzyme [Yon (1972) Biochem. J. 128, 311-320], the half-saturating concentrations of UMP and of the first substrate, carbamoyl phosphate (but not of the second, L-aspartate), were found to be strongly interdependent. However, the kinetic behaviour of the pure enzyme differed from that of the crude enzyme in several important respects, namely: (a) the apparent affinity for UMP was lower with the pure enzyme; (b) sigmoidicity was absent from plots of initial rate versus carbamoyl phosphate concentration, each at a fixed UMP concentration; (c) sigmoidicity was greatly exaggerated in plots of initial rate versus UMP concentration, each at a fixed carbamoyl phosphate concentration, owing to the occurrence of a slight but definite maximum in each plot at low UMP concentration; (d) there was a relative increase in this maximum in the presence of N-phosphonacetyl-L-aspartate, an inhibitor competitive with carbamoyl phosphate. It is shown that a modified two-conformation concerted-transition model can be used to account for most of these features of the pure enzyme. The model treats carbamoyl phosphate and UMP as antagonistic allosteric ligands binding to alternative conformational states [Monod, Wyman & Changeux (1965) J. Mol. Biol. 12, 88-118], carbamoyl phosphate binding non-exclusively (dissociation constants 20 microM and 85 microM respectively) and UMP binding exclusively (dissociation constant 2.5 microM). The model postulates further that the conformation with lower affinity for carbamoyl phosphate has the higher value of kcat., and that it binds UMP in competition with carbamoyl phosphate. Parameters giving the best fit of experimental data to this model were found by a non-linear least-squares search procedure.     

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.     

An in vitro model for the study of hydroponic forcing in chicory (Cichorium intybus)

An in vitro model for studying the influence of different factors on chicon formation during hydroponic forcing has been developed. The shoot apex was isolated from the chicory roots and cultured on a gelled nutrient medium. This medium was considered as a replacement of the root. Small chicons (5 g) were produced. Water and, more importantly, sucrose availability had important influences on the outgrowth of the chicons. When sucrose was added to the medium the chicon-weight increased two-fold. On a medium with low agar concentration (0.3% (w/v)), heavier chicons were produced compared with a medium with agar at 1.2% (w/v). Browning of the pith tissue (= flowering stem) decreased with increased agar concentration. The results presented indicate that the in vitro system can be used as a research model to study chicon development in relation to root functioning and composition. This revised version was published online in June 2006 with corrections to the Cover Date.     

A kinetic model of cooperativity in aspartate transcarbamylase.

A relatively simple kinetic model is proposed to account simultaneously for data on the binding of carbamyl phosphate and succinate to aspartate trans carbamylase (ATCase), and for the relaxation spectrum associated with this binding. The model also accounts for measurements of the initial velocity of the reaction of ATCase with respect to aspartate and carbamyl phosphate. The principal assumption made is that ATCase consists of three identical noninteracting cooperative dimers. Ordered binding and both sequential and concerted conformational changes in the dimers are needed to account for the properties of ATCase. The values of the parameters of this model can be determined by fitting to existing experimental evidence. Various new quantitative predictions are made that can serve as additional tests of the proposed theory.     

Regulation of the Pi-ATP exchange and hydrolytic reactions in F0-F1 reconstituted liposomes

The regulation of ATP hydrolysis and Pi-ATP exchange reactions by ATP, ADP, Mg2+, and phosphate was studied in liposomes containing F0-F1 obtained from bovine heart submitochondrial particles by solubilization with lauryl dimethylamino oxide as described previously (Dreyfus, G., Celis, H., and Ramirez, J. (1984) Anal. Biochem. 142, 215-220). A simultaneous analysis of ATP hydrolysis and the Pi-ATP exchange reactions showed that the ratio of hydrolysis/exchange is close to one when the ATP concentration is in the lower micromolar range. In this preparation ADP stimulates the Pi-ATP exchange reaction and depresses ATP hydrolysis. The exchange reaction is almost abolished when ADP is removed from the medium by an ATP-regenerating system. Mg2+ in millimolar concentrations stimulates Pi-ATP exchange, and at the same time decreases ATP hydrolysis; accordingly, the hydrolysis/exchange ratio depends on the concentration of Mg2+. Inorganic phosphate also controls this ratio, a lower ratio being observed at high phosphate concentrations. The Pi-ATP exchange reaction, but not ATP hydrolysis, depends on the concentration of medium phosphate. These results indicate that the kinetic characteristics of this F0-F1 preparation are modified by Mg2+, ATP, and phosphate.     

Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.     

Localization of d-myoinositol 1:2-cyclic phosphate 2-phosphohydrolase in rat kidney

1. On subcellular fractionation of rat kidney homogenates by differential and density-gradient centrifugation, the bulk of the inositol 1:2-cyclic phosphate 2-phosphohydrolase activity remains with the alkaline phosphatase activity, suggesting localization in the brush borders of the proximal tubules. 2. Histochemical studies with a medium containing inositol 1:2-cyclic phosphate and Escherichia coli phosphomonoesterase show Gomori staining around the brush borders of the proximal tubules in the outer cortex only. 3. Serial sections across the kidney from cortex perimeter to papilla suggest that the inositol 1:2-cyclic phosphate 2-phosphohydrolase has a limited distribution along the proximal tubule of the nephron, probably being limited to the pars convoluta, whereas the alkaline phosphatase extends along the pars recta.     

Oxygen balance for small organisms: an analytical model

An analytical model is developed that describes oxygen transport and oxygen consumption for small biological structures without a circulatory system. Oxygen inside the organism is transported by diffusion alone. Oxygen transfer towards the organism is retarded by a thin static fluid film at the surface of the organism. The thickness of this film models the outward water conditions, which may range from completely stagnant water conditions to so-called well-stirred water conditions. Oxygen consumption is concentration-independent above a specified threshold concentration (regulator behaviour) and is proportional to the oxygen concentration below this threshold (conformer behaviour). The model takes into account shape and size of the organism and predicts the transition from (pure) regulator behaviour to (pure) conformer behaviour, as well as the mean oxygen consumption rate. Thereby the model facilitates a proper analysis of the physical constraints set on shape and size of organisms without an active internal oxygen transport mechanism. This analysis is carried out in some detail for six characteristic shapes (infinite sheet, cylinder and beam; finite cylinder, sphere and block). In a well-stirred external medium, a flattened shape appears to be the most favourable for oxygen supply, while a compact shape (cube) is more favourable if the external medium is nearly stagnant. The theoretical framework is applied to oxygen consumption data of eight teleost embryos. This reveals relative insensitivity to external flow conditions in some species (e.g., winter flounder, herring), while others appear to rely on external stirring for a proper oxygen supply (e.g., largemouth bass). Interestingly, largemouth bass is the only species in our analysis that exhibits ‘fin-fanning’.     

Kinetic characterization of the two phosphate uptake systems in the fungus Neurospora crassa.

The kinetics of phosphate uptake by exponentially growing Neurospora crassa were studied to determine the nature of the differences in uptake activity associated with growth at different external phosphate concentrations. Conidia, grown in liquid medium containing either 10 mM or 50 micronM phosphate, were harvested, and their phosphate uptake ability was measured. Initial experiments, where uptake was examined over a narrow concentration range near that of the growth medium, indicated the presence of a low-affintiy (high Km) system in germlings from 50 micronM phosphate. Uptake by each system was energy dependent and sensitive to inhibitors of membrane function. No efflux of phosphate or phosphorus-containing compounds could be detected. When examined over a wide concentration range, uptake was consistent with the simultaneous operation of low- and high-affinity systems in both types of germlings. The Vmax estimates for the two systems were higher in germlings from 50 micronM phosphate than for the corresponding systems in germlings from 10 mM phosphate. The Km of the high-affinity system was the same in both types of germlings, whereas the Km of the low-affinity system in germlings from 10 mM phosphate was about three three times that of the system in germlings from 50 micronM phosphate.     

Competitive lipase-catalyzed ester hydrolysis and ammoniolysis in organic solvents; equilibrium model of a solid-liquid-vapor system.

Enzymatic ester hydrolysis and ammoniolysis were performed as competitive reactions in methyl isobutyl ketone without a separate aqueous phase. The reaction system contained solid ammonium bicarbonate, which dissolved as water, ammonia, and carbon dioxide. During the reaction an organic liquid phase, a vapor phase, and at least one solid phase are present. The overall equilibrium composition of this multiphase system is a complex function of the reaction equilibria and several phase equilibria. To gain a quantitative understanding of this system a mathematical model was developed and evaluated. The model is based on the mass balances for a closed batch system and straightforward relations for the reaction equilibria and the solubility equilibria of ammonium bicarbonate, the fatty acid ammonium salt, water, ammonia, and carbon dioxide. For butyl butyrate as a model ester and Candida antarctica lipase B as the biocatalyst this equilibrium model describes the experiments satisfactorily. The model predicts that high equilibrium yields of butyric acid can be achieved only in the absence of ammoniolysis or in the presence of a separate water phase. However, high yields of butyramide should be possible if the water concentration is fixed at a low level and a more suited source of ammonia is applied.     

Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies.

The parallel plate flow chamber provides a controlled environment for determinations of the shear stress at which cells in suspension can bind to endothelial cell monolayers. By decreasing the flow rate of cell-containing media over the monolayer and assessing the number of cells bound at each wall shear stress, the relationship between shear force and binding efficiency can be determined. The rate of binding should depend on the delivery of cells to the surface as well as the intrinsic cell-surface interactions; thus, only if the cell flux to the surface is known can the resulting binding curves be interpreted correctly. We present the development and validation of a mathematical model based on the sedimentation rate and velocity profile in the chamber for the delivery of cells from a flowing suspension to the chamber surface. Our results show that the flux depends on the bulk cell concentration, the distance from the entrance point, and the flow rate of the cell-containing medium. The model was then used in a normalization procedure for experiments in which T cells attach to TNF-alpha-stimulated HUVEC monolayers, showing that a threshold for adhesion occurs at a shear stress of about 3 dyn/cm2.