Transport experiments

For membrane transport experiments, the relevant membrane is sandwiched between two solutions a donor typicaUy at constant dmg concentration, C = Cg, and a receiver at zero concentration, C = 0. The dmg concentration in the receiver is monitored as a function of time and the cumulative amount transported, has a linear asymptote with time where M is the area,/ is the steady-state flux, /is the time, and / is the time lag. 

We will discuss below the reeent experimental observations relative to the eleetrieal resistivity and magnetoresistance of individual and bundles of MWCNTs. It is interesting to note however that the ideal transport experiment, i.e., a measurement on a well eharacterised SWCNT at the atomic scale, though this is nowadays within reaeh. Nonetheless, with time the measurements performed tended gradually eloser to these ideal eonditions. Indeed, in order to interpret quantitatively the eleetronie properties of CNTs, one must eombine theoretieal studies with the synthesis of well defined samples, which structural parameters have been precisely determined, and direet electrical measurements on the same sample. 

Blending NBR with ENR improves physical and mechanical properties without affecting its oil resistance. Such blends can be used for making per-evaporation membranes. Mathai et al. have studied the transport properties of aromatic solvents through membranes prepared from 50 50 NBR-ENR blends [36]. Transport experiments were carried out by immersing circular specimens in the desired... 

Patrone, L., Paladn, S., CharHer, J., Armand, F., Bourgoin, J.P., Tang, H. and Gauthier, S. (2003) Evidence of ffie Key Role of Metal-Molecule Bonding in Metal-Molecule-Metal Transport Experiments. Physical Review Letters, 91, 096802. 

Comparisons with Other Hole Transport Experiments... 

Rigorous calibration is a requirement for the use of the side-by-side membrane diffusion cell for its intended purpose. The diffusion layer thickness, h, is dependent on hydrodynamic conditions, the system geometry, the spatial configuration of the stirrer apparatus relative to the plane of diffusion, the viscosity of the medium, and temperature. Failure to understand the effects of these factors on the mass transport rate confounds the interpretation of the data resulting from the mass transport experiments. 

In principle, this method would also be useful for transport experiments in which the drug is dissolved rather than suspended in the semisolid. Since the stirring rate can be varied, this system provides for studying stirring rate dependence. An additional advantage is that the transport results are not complicated by the use of a membrane to separate the donor and receptor phases. 

In the selection of an appropriate cell culture system, a number of criteria must be considered (Table 3). These include not only the characteristics of the cell type but also the controllable parameters of the complete transport system such as the permeants, the filter properties, and the assay conditions. In general, most transport experiments employ the experimental design shown schematically in Figure 4 with modifications as discussed below. Typically, the desired cell is seeded onto some sort of semipermeable filter support and allowed to reach confluence. The filter containing the cell monolayer separates the donor and receiver... 

Several examples have already been pointed out in which the properties of the solute itself can impact on the results obtained from a transport experiment. Metabolic instability and propensity for nonspecific adsorption are problems which can frequently be encountered and must be considered any time a new solute is to be studied. In addition to these problems, there are several other solute-related factors which must be considered in the design and interpretation of transport studies. 

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

Typically, transport experiments have been performed using a U-tube apparatus in which a solvent such as chloroform, containing the macrocyc-lic carrier, is placed in the tube so that it separates two aqueous phases the source phase containing the metal ion(s) to be transported and the receiving phase into which the transported ions are deposited. A diagrammatic representation of a liquid membrane system is shown in Figure 9.4. 

It should be noted that selective carrier facilitated transport experiments involving the crown ethers and their derivatives have not been... 

Such an experimental characterization is a necessary step to carry out a detailed comparison of emission properties as measured experimentally with the corresponding quantities as calculated by numerical models capable of describing transport and energy deposition of fast electrons in matter and consequent emission of characteristic X-ray emission. A possible modeling approach of fast electron transport experiments is given here, where the above results on Ka imaging were interpreted using the hybrid code PETRA [53] to... 

Pedley, T. J. (1983). Calculations of unstirred layer thickness in membrane transport experiments a survey, Q. Rev. Biophys., 16, 115-150. 

With the larger racemic cr-hydroxy-l-naphthaleneacetic acid too, extraction takes place in the presence of 1309] and the appropriate cations but enantiomeric differentiation is not observed. These results were confirmed in transport experiments in which the alkali mandelate is carried through a liquid membrane of [309] dissolved in chloroform. Lehn et al. (1978) explain these observations in terms of an ion pair included in the cavity of the crown ether. The reversal of chiral recognitions between potassium and cesium mandelate of 25% indicates that the structures of the two complexes are different. 

An alternative method for assessing cell layer integrity is through the use of hydrophilic paracellular transport markers (e.g., radiolabeled D-mannitol or fluorescein-Na+), which passively traverse cells by the paracellular route. Small amounts of compound required for in vitro conjunctival cell culture transport experiments make this approach well suited for screening purposes. Relative absorption index of a series of pharmacologically active molecules can be ranked against known markers for the identification of candidates with potential absorption problems, which is a reliable tool to select drug candidates with optimal characteristics. 

Either Transwell inserts or side-by-side diffusion chambers can be used for transport studies. Bode et al. have provided an excellent review on this subject [60], Briefly, cells are incubated for 30-60 min with a buffer solution. To initiate the transport study, a transport buffer containing the drug under investigation is added to either the apical or the basal chamber depending on the transport direction of interest. At predetermined time points, the respective receiver chamber is sampled and the withdrawn volume is replaced with the same volume of fresh buffer. The permeability coefficient (Papp) is calculated and the ratio of /apP in the basolateral-to-apical direction versus that in the apical-to-basolateral direction gives the efflux ratio. These sort of transport experiments are well suited to determine if drugs/xenobiotics are substrates of the placental efflux proteins. 

The intention to study transport processes at pulmonary epithelia, however, raised two particular problems (i) the apical side of these epithelia is typically in contact with air rather than with a liquid and (ii) in order to maximize the surface area, the lungs have a complex treelike structure, ending in millions of tiny alveolar bubbles. The total surface area of the human alveolar epithelium is almost half of that of the intestines (100-120 m2), with its macroscopic appearance resembling a sponge, and it is virtually impossible to use such a tissue for transport experiments in a diffusion-chamber setup. 

Compared to in vivo studies, the Caco-2 model substantially increases the speed at which absorption potential can be estimated and reduces the amount of drug substance needed. However, manually performed assays are still too slow and labor intensive compared to biological high-throughput screening assays. Caco-2 cells take about 3 weeks to form monolayers of fully differentiated cells. At this point, Caco-2 monolayers are used to evaluate absorption potential under a variety of permeability protocols. In order to further expedite the process of absorption potential assessment, efforts have been made to increase the throughput of Caco-2 transport experiments. 

Nickel availability to the host plants severely limits the expression of the R. leguminosarum hydrogenase genes in the P. sativum symbiosis (Brito et al. 1994) and probably in other symbioses such as M. loti-L. corniculatus (Brito et al. 2000). This limitation occurs at the level of processing of the enzyme subunits (Brito et al. 1994). It is not clear, however, whether Ni limitation is due to the bacterial or the plant component of the symbiosis. Recent results of nickel transport experiments with intact pea symbiosomes indicate that the peribacteroid membrane is not a specific barrier for Ni transport into the bacteroid (Bascones et al. unpublished). 

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