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PubMed2006

Cell lysis inside the capillary facilitated by transverse diffusion of laminar flow profiles (TDLFP).

Chemical cytometry studies the molecular composition of individual cells by means of capillary electrophoresis or capillary chromatography. In one of its realizations an intact cell is injected inside the capillary, the plasma membrane is disrupted to release the cellular contents into the separation buffer, and, finally, the molecules of interest are separated and detected. The solubilization of the plasma membrane with a surfactant is a simple and efficient way of achieving cell lysis inside the capillary. To facilitate cell lysis by a surfactant the cell has to be contacted with the surfactant inside the capillary. We recently introduced a generic method for mixing solutions inside the capillary termed transverse diffusion of laminar flow profiles (TDLFP). In this work, we propose that TDLFP can facilitate efficient cell lysis inside the capillary. Conceptually, a short plug of the surfactant is injected by pressure prior to cell injection. The cell is then injected by pressure within a plug of the physiological buffer. Due to the parabolic profiles of pressure-driven laminar flows the interface between the plug of the surfactant and that of the physiological buffer is predominantly longitudinal. Transverse diffusion mixes the surfactant with the physiological buffer, which leads to surfactant's contact with the cell and subsequent cell lysis. Here, we demonstrate that the proposed concept is valid. TDLFP-facilitated cell lysis by a short plug of the surfactant allows us to exclude the surfactant from the run buffer, and, hence, facilitates modes of separation, which are incompatible with the surfactant's presence in the run buffer. In addition to cell lysis, TDLFP will be used to mix the cellular components with labeling reactants, affinity probes, inhibitors, etc. We foresee that the generic nature and enabling capabilities of TDLFP will speed up the maturation of chemical cytometry into a practical bioanalytical tool.

Animals↗
PubMed2000

Diffusion of nitric oxide can facilitate cerebellar learning: A simulation study.

The gaseous second messenger nitric oxide (NO), which readily diffuses in brain tissue, has been implicated in cerebellar long-term depression (LTD), a form of synaptic plasticity thought to be involved in cerebellar learning. Can NO diffusion facilitate cerebellar learning? The inferior olive (IO) cells, which provide the error signals necessary for modifying the granule cell-Purkinje cell (PC) synapses by LTD, fire at ultra-low firing rates in vivo, rarely more than 2-4 spikes within a second. In this paper, we show that NO diffusion can improve the transmission of sporadic IO error signals to PCs within cerebellar cortical functional units, or microzones. To relate NO diffusion to adaptive behavior, we add NO diffusion and a "volumic" LTD learning rule, i.e., a learning rule that depends both on the synaptic activity and on the NO concentration at the synapse, to a cerebellar model for arm movement control. Our results show that biologically plausible diffusion leads to an increase in information transfer of the error signals to the PCs when the IO firing rate is ultra-low. This, in turn, enhances cerebellar learning as shown by improved performance in an arm-reaching task.

Action Potentials↗
PubMed1988

Oxygen transfer of red blood cells: experimental data and model analysis.

Kinetics of O2 uptake and release by human red blood cells (RBC) as measured by stopped-flow techniques were simulated using an RBC model shaped as a spheric shell. The O2 transfer mechanisms in this model include diffusion and reaction within the RBC and diffusion and convection in the medium surrounding the RBC. Unknown model parameters were determined by comparing simulations with experimental data. The following conclusions were drawn. (1) Both diffusion and convection contribute to O2 transport in the medium surrounding the RBC, and this transport importantly limits the overall O2 transfer kinetics in stopped-flow experiments. (2) Intraerythrocyte transport mechanisms become predominant in limiting O2 transfer, and can thus be investigated by stopped-flow techniques, only when the perierythrocyte O2 transport resistance is minimized, e.g. by high levels of dithionite in measurements of O2 release from RBC. (3) Intraerythrocyte O2 transfer is shown to be mainly limited by diffusion of O2 and, to a lesser extent, by diffusion of oxyhemoglobin ('facilitated O2 diffusion') and by O2/hemoglobin reaction. The results suggest that diffusion is the main process limiting O2 uptake and release by RBC, the finite reaction kinetics of O2 with hemoglobin exerting a smaller limiting effect.

Erythrocytes↗
PubMed1995

Calbindin-D28K facilitates cytosolic calcium diffusion without interfering with calcium signaling.

The role of calbindin-D28K, in transcellular Ca2+ transport and Ca2+ signaling in rabbit cortical collecting system was investigated. Rabbit kidney connecting tubules and cortical collecting ducts, hereafter referred to as cortical collecting system, were isolated by immunodissection and cultured to confluence on permeable filters and glass coverslips. Calbindin-D28K was present in the cytosol of principal cells, but was absent from the intercalated cells. 1,25(OH)2D3 (48 h, 10(-7) M) significantly increased cellular calbindin-D28K levels (194 +/- 15%) and stimulated transcellular Ca2+ transport (41 +/- 3%). This stimulatory effect could be fully mimicked by the endogenous Ca2+ chelator, BAPTA (30 microM BAPTA/AM), which suggests that the presence of Ca2+ chelators alone is sufficient to enhance transcellular Ca2+ transport. Stimulation of Ca2+ transport was not accompanied by a rise in [Ca2+]i. Isosmotic replacement of extracellular Na+ ([Na+]o) for N-methylglucamine (NMG) generated oscillations in [Ca2+]i in individual cells of the monolayer. The functional parameters of these oscillations such as frequency of spiking, resting [Ca2+]i, increase in [Ca2+]i and percentage of responding cells, were not affected by the level of calbindin-D28K. In contrast, loading the cells with BAPTA abruptly stopped these [Ca2+]i oscillations. This suggests that the kinetics of Ca2+ binding by calbindin-D28K are slow relative to the initiation of the [Ca2+]i rise, so that calbindin-D28K, unlike BAPTA, is unable to reduce [Ca2+]i rapidly enough to prevent the initiation of Ca(2+)-induced Ca2+ release.

Animals↗
PubMed2001

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells.

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, D(r) (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, D(l) (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22 degrees C, D(l) for myoglobin is 1.2 x 10(-7) cm(2)/s in soleus fibers and 1.1 x 10(-7) cm(2)/s in cardiomyocytes. D(l) for lactalbumin is similar in both cell types. D(r) for myoglobin is 1.2 x 10(-7) cm(2)/s in soleus fibers and 1.1 x 10(-7) cm(2)/s in cardiomyocytes and, again, similar for lactalbumin. D(l) and D(r) for ovalbumin are 0.5 x 10(-7) cm(2)/s. In the case of myoglobin, both D(l) and D(r) at 37 degrees C are about 80% higher than at 22 degrees C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, approximately 1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Animals↗
PubMed1976

Measurements of exit rates to distinguish between facilitated and simple diffusion.

A modification of the method of Sen and Widdas (J. Physiol. London 160:392-403, 1962) was used to measure the rate of exit of several nonelectrolytes from erythrocytes of various species. In spite of additional errors introduced by the larger half-saturation values of the carriers (phi) and concentrations, it was possible to distinguish between systems with small values of phi, systems with relatively large values of phi, and systems involving only simple diffusion. Approximate values of phi in millimoles and of maximum transfer rate (K) in isotonic units per minute were obtained using times and initial slopes measured on experimental curves. The following values in the foregoing units were obtained: human glucose, phi = 1.8, 1.0, K = 0.8, 1.1; human glycerol, phi = 178, 94, K = 4.3, 3.3; sheep thiourea, phi = 56, 56, K = 0.9, 0.6; and rabbit glycerol, phi = 328, 64, K = 2.2, 1.0. Simple diffusion was demonstrated for the following systems: ox-ethylene glycol; ox-glycerol; sheep-ethylene glycol; and sheep glycerol.

Animals↗
PubMed1975

Anti-immunoglobulin analysis by diffusion patterns of inhibition and facilitation of complementary lysis in agar. II. Diffusion-lysis as a method for recognizing in vivo immunosuppressive activity of anti-immunoglobulins.

This paper provides evidence that it is possible to prepare facilitating anti-mouse immunoglobulin (that is, anti-mouse immunoglobulin which facilitates complementary lysis of red cells sensitized with mouse-produced haemolysin) which, when injected into mice 24 hours before an injection of sheep red cells, very markedly reduced the number of haemolysin-producing cells detectable in spleen four days later. The diffusion-lysis method was used to recognize this and other anti-Ig's in heterologous antiserum and fractions thereof. The effective antibody was in the gamma2 fraction of antiserum produced in guinea pigs by injecting them with guinea pig red cells sensitized with mouse-produced haemolysin. This method of immunizing was used in order to stimulate the production of antibody against immunoglobulin which had undergone the configurational change characteristically occurring when antibody unites with antigen. The 19S fraction of the antiserum contained inhibiting anti-mouse immunoglobulin (anti-mouse immunoglobulin which inhibits complementary lysis of red cells sensitized with mouse-produced haemolysin) and interfered with immune depression by the gamma2 fraction. It is postulated that the gamma2 fraction induces complementary lysis only of lymphocytes whose surface immunoglobulin receptors have bound antigen and undergone configurational change. It is suggested that facilitating anti-immunoglobulin of the type described is responsible for immune suppression by anti-lymphocyte serum (ALS). Facilitating anti-mouse immunoglobulin was demonstrated in two samples of ALS (anti-mouse) which were active in suppressing graft rejection, but inhibiting anti-mouse immunoglobulin only was found in a sample which was ineffective in suppressing graft rejection.

Agar↗
PubMed2006

Myoglobin translational diffusion in rat myocardium and its implication on intracellular oxygen transport.

Current theory of respiratory control invokes a role of myoglobin (Mb)-facilitated O2 diffusion in regulating the intracellular O2 flux, provided Mb diffusion can compete effectively with free O2 diffusion. Pulsed-field gradient NMR methods have now followed gradient-dependent changes in the distinct 1H NMR gamma CH3 Val E11 signal of MbO2 in perfused rat myocardium to obtain the endogenous Mb translational diffusion coefficient (D(Mb)) of 4.24 x 10(-7) cm2 s(-1) at 22 degrees C. The D(Mb) matches precisely the value predicted by in vivo NMR rotational diffusion measurements of Mb and shows no orientation preference. Given values in the literature for the Krogh's free O2 diffusion coefficient (K0), myocardial Mb concentration and a partial pressure of O2 that half saturates Mb (P50), the analysis yields an equipoise diffusion P(O2) of 1.77 mmHg, where Mb and free O2 contribute equally to the O2 flux. In the myocardium, Mb-facilitated O2 diffusion contributes increasingly more than free O2 diffusion when the P(O2) falls below 1.77 mmHg. In skeletal muscle, the P(O2) must fall below 5.72 mmHg. Altering the Mb P50 induces modest change. Mb-facilitated diffusion has a higher poise in skeletal muscle than in myocardium. Because the basal P(O2) hovers around 10 mmHg, Mb does not have a predominant role in facilitating O2 transport in myocardium but contributes significantly only when cellular oxygen falls below the equipoise diffusion P(O2).

Adenosine Triphosphate↗
PubMed1975

Anti-immunoglobulin analysis by diffusion patterns of inhibition and facilitation of complementary lysis in agar. I. Diffusion-lysis patterns of heterologous guinea pig anti-immunoglobulins.

Anti-rabbit immunoglobulin-containing sera were produced by immunizing guinea pigs with guinea pig red cells sensitized with rabbit-produced haemolysin against them. Such sera, and their 7S and 19S fractions, were radially diffused in agar containing sheep red cells sensitized with rabbit haemolysin and fractions thereof. After diffusion, the preparations were treated with guinea pig complement, which produced partial lysis in the background and a series of alternating rings of more and less lysis than that in the background. These were interpreted as a due to the presence of at least four anti-immunoglobulins (anti-Ig's) as follows: a 19S anti-Ig which inhibited lysis by 7S and 19S haemolysin; a 7S anti-Ig which facilitated lysis by 7S and 19S heamolysin; a 7S anti-Ig which inhibited lysis by 7S and 19S haemolysin; a 7S anti-Ig which facilitated lysis by 19S haemolysin. Similar patterns were obtained using correspondingly produced sera against mouse immunoglobulin, and sensitizing the sheep cells in the agar with mouse-produced haemolysin.

Animals↗
PubMedDate not supplied

Studies of ligand diffusion pathways over a protein surface.

Studies were conducted on the behavior of simulated molecules diffusing within organized water, postulated to form from the hydropathic states of protein surface amino acid side chains. This organization is postulated to facilitate the diffusion of ligands across the protein surface to their effector. These studies reveal that the organized water can be disrupted in their diffusion facilitating function by the presence of some other solute in high concentration. It was also found from cellular automata simulations that chiral isomers behaved in a slightly different manner when in an asymmetric enclosure simulating a fragment of the organized water pathway. These findings have relevance to observations about the mechanism of action of nonspecific anesthetic agents.

Amino Acids↗
PubMed1993

Transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea in the human glioma cell line SK-MG-1 is mediated by an epinephrine-sensitive carrier system.

The transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental anticancer compound, was investigated in the human glioma cell line SK-MG-1. The transport of [3H]SarCNU was examined in suspension. The uptake of [3H] SarCNU was found to be temperature dependent, with influx being linear to 4 sec at 37 degrees. Equilibrium was reached after 1 min at 22 degrees and 37 degrees, with accumulation slightly above unity. SarCNU was not significantly metabolized in the cells after a 60-min incubation at 37 degrees, as shown by thin layer chromatography. At 37 degrees, uptake of [3H]SarCNU was found to be saturable, sodium independent, and energy independent. Previous work demonstrated that SarCNU was able to inhibit the uptake of sarcosinamide, which is transported by the catecholamine uptake 2 system. This catecholamine system mediates the physiological transport of epinephrine. Epinephrine was able to significantly inhibit the uptake of [3H]SarCNU, at a concentration of 50 microM, by 40%. Additionally, several amino acids were unable to inhibit the uptake of SarCNU. The initial rate of SarCNU influx is mediated by both facilitated and nonfacilitated diffusion. The nonfacilitated diffusion rate could be estimated from the linear concentration dependence of the residual influx rate for SarCNU, which was not inhibited by the presence of excess co-permeant (epinephrine). Dixon plot analysis, corrected for nonfacilitated diffusion of SarCNU, revealed that epinephrine inhibited the uptake of SarCNU competitively, with a Ki of 163 +/- 15 microM, a value similar to the Km value for epinephrine influx in SK-MG-1 cells. Additionally, after appropriate corrections for nonfacilitated diffusion in the influx rates observed for SarCNU, it was revealed that SarCNU influx obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations, with a Km of 2.39 +/- 0.37 mM and a Vmax of 236 +/- 53 pmol/microliters of intracellular water/sec. Metabolic poisons (2,4-dinitrophenol, iodoacetate, NaCN, NaF, or ouabain) were unable to inhibit the influx of SarCNU, suggesting that the carrier-mediated uptake of SarCNU is energy independent and mediated by facilitated diffusion. These findings indicate that SarCNU uptake in SK-MG-1 cells is mediated both by nonfacilitated diffusion and by facilitated diffusion via the catecholamine uptake 2 carrier system. SarCNU is the first chloroethylnitrosourea that has been demonstrated to have carrier-mediated uptake. Moreover, this carrier-mediated uptake may play a role in the increased cytotoxicity of SarCNU against gliomas, compared with that of 1,3-bis(2,-chloroethyl)-1-nitrosourea, which enters cells primarily by passive diffusion.

Antineoplastic Agents↗
PubMed1999

Production and oxidation of wool grease after shearing.

OBJECTIVE: To measure the production and amount of oxidation of wool grease secreted immediately after shearing. To identify components of wool grease that might act as a carrier to facilitate lateral diffusion of topically applied insecticides. DESIGN: Fine-wool Merino sheep were shorn and residual greasy wool was collected from the sheep's flank. The quantity of grease produced, and the amount of oxidation was measured during 18 days after shearing. Wool grease was fractionated into five component groups based on their polarity and the degree of oxidation in these fractions determined. RESULTS: There was a 24% increase in grease production within 2 days after shearing but secretions returned to pre-shearing amounts after 4 days. During this period wool grease oxidized rapidly. Of the grease fractions examined, sterol and wax esters remained essentially unoxidized whereas free sterols such as cholesterol and lanosterol, fatty acids and polar lipids, aldehydes and alcohols were extensively oxidized within 7 days after shearing. CONCLUSION: The transient increase in grease production after shearing may facilitate diffusion of topically applied synthetic pyrethroid insecticides. Oxidation of grease components may then contain the insecticide and limit further diffusion. Incorporating the insecticide in non-oxidising fractions of wool grease may make insecticide dispersion more efficient.

Administration, Cutaneous↗
PubMed1990

Calculated intra- and extracellular PO2 gradients in heavily working red muscle.

A recently introduced three-dimensional analytical model of O2 diffusion to heavily working muscle that considers myoglobin-facilitated O2 diffusion inside the muscle fiber and a carrier-free layer separating erythrocytes and fiber is able to furnish the following new insights in O2 supply to red muscle at high performance. 1) Fiber PO2 profiles are essentially flat, and the major PO2 gradients are located in the perierythrocytic region, in good agreement with experimental findings [T. E. J. Gayeski and C. R. Honig, Am. J. Physiol. 251 (Heart Circ. Physiol. 20): H789-H799, 1986]. No specialized anatomical pericapillary barrier structure is required to explain these results. 2) A functional barrier to O2 diffusion has been identified that consists of the carrier-free layer and of the pericapillary muscle fiber portions. There are three reasons that make these structures act as a diffusion barrier: a "geometric reason," a "diffusivity-related reason," and a "myoglobin-related reason." 3) PO2 fields of adjacent red blood cells (RBCs) practically do not interact. 4) Small scale heterogeneities in capillary and RBC spacing are compensated for by high myoglobin-facilitated fiber diffusivity. Limiting factor for diffusional O2 transport is the number of RBCs present on the fiber surface.

Animals↗
PubMed1995

An easy-to-use model for O2 supply to red muscle. Validity of assumptions, sensitivity to errors in data.

An easy-to-use capillary cylinder model of O2 supply to muscle is presented that considers all those factors that are known to be most important for realistic results: (1) red blood cell (RBC) O2 unloading along the capillary, (2) effects of the particulate nature of blood, (3) free and hemoglobin-facilitated O2 diffusion and reaction kinetics inside RBCs, (4) free and myoglobin-facilitated O2 diffusion inside the muscle cell, and (5) carrier-free region separating RBC and tissue. In a first approach, a highly simplified yet reasonably accurate treatment of the complex three-dimensional oxygen diffusion field in and next to capillaries is employed. As an alternative, a more realistic description using RBC/capillary diffusing capacity has been included. Model development proceeds step by step and is designed to be easily comprehensible for a broad readership. In spite of the number of features accounted for, the model is simple to apply, even for scientists not specialized in the field of modeling. PO2 distributions calculated by the model are in good qualitative agreement with experimental data and with former modelling results. By means of suitable extensions to the model that are also developed it is shown for a wide range of muscle performances that quite generally the following complication may be neglected safely: (1) complexity of O2 diffusion field near capillaries, (2) deviations of capillary domain cross sections from the circular shape, (3) O2 diffusion parallel to the capillary direction, and (4) PO2 dependence of O2 consumption rate. Finally, a sensitivity analysis is performed in which propagation of errors in the input data into the results is investigated. The interpretation of the calculated sensitivities gives insights in the specific dependencies of muscular O2 supply on the various input parameters. Moreover, basic interrelations governing carrier-facilitated diffusional O2 transport to muscle become apparent and are discussed.

Animals↗
PubMedDate not supplied

[Mechanisms of absorption of amino acids and oligopeptides. Control and implications in human diet therapy].

The mechanisms involved in the absorption of amino acids and oligopeptides are reviewed regarding their implications in human feedings. Brush border and basolateral membranes are crossed by amino acids and di-tripeptides by passive (facilitated or simple diffusion) or active (Na+ or H+ co-transporters) pathways. Active Na(+)-dependent system occurs mainly at brush border and simple diffusion at basolateral, both membranes have the passive facilitated transport. Free-amino acids use either passive or active transport systems whereas di-tripeptides do mainly active (H+ co-transporter). Brush border have distinctive transport system for amino acids and di-tripeptides. The former occurs mainly by active Na+ dependently whereas the later is active H(+)-dependent with little affinity for tetra or higher peptides. Free amino acids are transported at different speed by saturable, competitive carriers with specificity for basic, acidic or neutral amino acids. Di and tripeptides have at least two carriers both electrogenic and H(+)-dependent. The basolateral membrane transport of amino acids is mostly by facilitated diffusion while for di-tripeptides it is an active anion exchange associated process. The main regulation of amino acids and di-tripeptide transport is the presence o substrate at the mucosal membrane with higher the substrate higher the absorption. Di and tripeptides are more efficiently absorbed than free amino acids which in turns are better absorbed than oligopeptides. So di-tripeptides result in better N-retention and is particularly useful in cases of lower intestinal absorption capacity. The non-absorbed peptides are digested and fermented by colonic bacteria resulting short-chain fatty acids, dicarboxylic acids, phenolic compounds and ammonia. Short-chain fatty acid provides energy for colonocytes and bacteria and the ammonia not fixed by bacteria returns to the liver for ureagenesis.

Amino Acids↗
PubMed2000

Facilitated uptake of fentanyl, but not alfentanil, by human pulmonary endothelial cells.

BACKGROUND: Extensive pulmonary uptake of lipophilic basic amines, such as fentanyl, attenuates early blood drug concentrations after rapid intravenous administration. The basis of this phenomenon is poorly understood. The authors tested the hypothesis that fentanyl uptake into cultured human lung microvascular endothelial (HMVE-L) cells occurs by facilitated uptake in addition to passive diffusion. The authors compared fentanyl and alfentanil uptake with that of antipyrine, a diffusible marker of pulmonary tissue water. In addition, the authors determined the effect of verapamil, a nonspecific inhibitor of drug transport, and UIC2, a blocking antibody of the P-glycoprotein drug transporter, on the uptake of these drugs. METHODS: Human lung microvascular endothelial cells were incubated, with varying concentrations of antipyrine and fentanyl or alfentanil in the absence or presence of varying verapamil concentrations or of UIC2. Supernatants were collected and cells were rinsed and dissolved. Supernatant and cell-associated antipyrine, fentanyl, and alfentanil concentrations were measured. The data were fit to a model of cellular uptake that allowed for passive diffusion and facilitated uptake. RESULTS: Alfentanil uptake by HMVE-L cells was indistinguishable from that of antipyrine for the concentration ranges studied. In contrast, at low concentrations, fentanyl sequestration into HMVE-L cells was substantially greater than that of antipyrine. Facilitated fentanyl uptake was blocked by verapamil, but not by UIC2, in a concentration-dependent manner. CONCLUSIONS: The differential HMVE-L uptake of fentanyl and alfentanil is consistent with the observed differences in the pulmonary uptake of these drugs. This suggests that specific fentanyl uptake and sequestration by HMVE-L cells may be the mechanisms of its extensive pulmonary uptake.

ATP Binding Cassette Transporter, Subfamily B, Mem↗