The combined use of the membrane surface potential fluorescent sensor fluorescein phosphatidylethanolamine (FPE) and the membrane dipole potential fluorescent sensor di-8-ANEPPS to characterize the interaction of molecules with model and cellular membranes and to asses the influence of the dipole potential on the interaction is reported. The study of the human immunodeficiency virus protease inhibitor saquinavir with Caco-2 cells and phospholipid membranes reveals that the compound interacts with the lipidic bilayer of model membranes with a simple hyperbolic binding profile but with Caco-2 cells in a cooperative way involving membrane receptors. Additional studies indicated that colchicine acts as a competitor ligand to saquinavir and suggests, in agreement with other reports, that the identity of the saquinavir "receptor" could be P-glycoprotein or the multiple drug resistance-associated protein. The modification of the magnitude of the membrane dipole potential using compounds such as cholesterol, phloretin, and 6-ketocholestanol influences the binding capacity of saquinavir. Furthermore, removal of cholesterol from the cell membrane using methyl-␤-cyclodextrin significantly decreases the binding capacity of saquinavir. Because removal of cholesterol from the cell membrane has been reported to disrupt membrane domains known as "rafts," our observations imply that the membrane dipole potential plays an important role as a modulator of molecule-membrane interactions in these membrane structures. Such a role is suggested to contribute to the altered behavior of receptor-mediated signaling systems in membrane rafts.The interactions between many types of differing molecules and biological membranes underlie much of the cell biology, physiology, and pathology. During the course of such interactions a number of physical factors play important roles and may be used to monitor such interactions. Three of the most influential parameters involve the different membrane potentials that have quite separate identities and origins but appear to be a feature of biological membranes (1). The membrane potentials include the transmembrane potential, resulting from a charge gradient across the membrane, the surface potential, arising from the net excess charge present at the membrane surface, and the membrane dipole potential, which has its origin in the molecular dipoles located on the membrane lipid molecules (1,7,41).

The objective of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) using a sonicated pure culture of Lawsonia intracellularis as the antigen (So-ELISA). A total of 332 serum samples, consisting of 232 experimentally infected animals and 100 animals naturally infected with L. intracellularis, were used to assess the diagnostic sensitivity. Three hundred and fifty-five sera from uninfected animals were used to determine the diagnostic specificity. The receiver operating characteristic and mean +3 standard deviation of optical density (OD) values from uninfected animals were used for selecting cut-off points. The diagnostic accuracy of So-ELISA was considered to be high as the area under the curve index was 0.991 with 0.0029 standard error. The optimal cut-off for So-ELISA was set at 0.45 OD with 89.8% sensitivity and 99.4% specificity based on a combination of good sensitivity and high specificity. No cross-reactivity was found in sera from pigs exposed to Brachyspira pilosicoli, B. hyodysenteriae, Campylobacter mucosalis, C. jejuni, or C. coli. Inter- and intracoefficient of variation of all control sera tested with So-ELISA was less than 10%. The observed agreements between So-ELISA and the immunoperoxidase monolayer assay tested with experimental challenge animals and field samples were 95.08% with 0.88 kappa and 90.65% with 0.74 kappa value, respectively. So-ELISA was able to detect the seroconversion of infected animals at 2 to 4 weeks after exposure to L. intracellularis. Based on the validation results, So-ELISA could be used as an alternative serology for proliferative enteropathy diagnosis.

Cats with IC have lower urinary GAG-to-creatinine concentration ratios than did clinically normal cats. Administration of NAG (250 mg, PO, q 24 h) significantly increased plasma GAG concentrations in cats with IC after 21 days of treatment.

The authors' laboratory has applied a series of different fluorescence assays for monitoring the binding and transport of fatty acids (FA) in model and biological membranes. The authors recently expanded their fluorescent assays for monitoring the adsorption of FA to membranes to a total of three probes that measure different aspects of FA binding: (1) an acrylodan-labeled FA-binding protein, which measures the partitioning of FA between membranes and the aqueous buffer; (2) the naturally occurring fluorescent cis-parinaric acid, which specifically measures the insertion of the FA acyl chain into the hydrophobic core of the phospholipid bilayer, and (3) a fluorescein-labeled phospholipid (N-fluorescein-5-thiocarbomoyl-1,2,dihexadecanoyl-sn-glycero-3-phosphoethanolamine), which specifically measures the arrival of the FA carboxyl at the outer leaflet of the membrane. None of these probes allow the transmembrane movement of FA to the inner leaflet to be measured. FA translocation (flip-flop) is typically measured directly, using a pH-sensitive fluorophore such as 8-hydroxypyrene-1.3.6-trisulfonic acid or 2',7'-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein. These probes detect the release of protons from unionized FA that have diffused through the membrane to the inner leaflet. Because adsorption of FA to the outer leaflet must occur before flip-flop, these probes measure the effects of the combined steps of adsorption and translocation. In this chapter, detailed methods are provided on how to monitor the transport of FA through protein-free model membranes, and some of the fluorescent artifacts that may arise with the use of these probes are addressed. Also, experiments designed to investigate such artifacts, and improve the reliability and interpretation of the data are described.