ABSTRACT
The retrograde transport of nerve growth factor (NGF) in neurite-like processes of living differentiated PC12 cells was studied using streptavidin-quantum dots (QDs) coupled to monobiotin-NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten-35% of the QD-NGF-receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step-like motion, requiring intact microtubules, with a net retrograde velocity of 0.054+/-0.020 microm/s. Individual runs had a mean velocity of approximately 0.15 microm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real-time analysis of individual QDbNGF- receptor complexes trafficking within neurites.
Subject(s)
Multiprotein Complexes/metabolism , Receptor, Nerve Growth Factor/metabolism , Staining and Labeling/methods , Animals , Chromogenic Compounds/pharmacokinetics , Endocytosis , Microtubules/physiology , Neurites/metabolism , Neurites/ultrastructure , PC12 Cells , Protein Binding , Protein Transport , Quantum Dots , Rats , Sensitivity and Specificity , Substrate SpecificityABSTRACT
There is an increasing interest in the role of coagulation factor XIII (FXIII) in cardio- and cerebrovascular diseases. It has recently been reported that a common G-->T point mutation in the A-subunit gene of FXIII, which codes for a valine (val) to leucine (leu) change (FXIIIVal34Leu), is protective against thrombotic diseases but seems to increase the risk of intracerebral bleeding. We developed a colorimetric incorporation assay for detection of FXIII activity based on incorporation of 5-(biotinamido) pentylamine (BAPA) into fibrin or fibrinogen. With this new assay, we studied the effects of FXIIIVal34Leu mutation, plasma fibrinogen concentration and congenital FXIII deficiency on FXIII activity. There are no data available about the ability of different FXIII assays to detect altered activity in FXIIIVal34Leu genotypes. We therefore compared our results determined by the incorporation method with a commonly used photometric method based on ammonia release after cross-linking of glycine-ethylester to a specific glutamine containing peptide substrate. We also determined FXIII A-subunit antigen (Ag) levels using enzyme-linked immunosorbent assay (ELISA) technique. The FXIIIVal34Leu genotype could not be detected either by the photometric method nor by the FXIII A-subunit ELISA. The incorporation assay showed an increased specific FXIII activity in subjects possessing the leu allele. The photometric assay and ELISA gave similar results independent from genotype. In patients with congenital FXIII deficiency before and after substitution, however, ELISA and the incorporation assay gave similar results, whereas the photometric assay showed consistently higher values. Our results show that the incorporation assay, not the photometric assay based on ammonia release, can be used for detection of elevated activity in subjects with FXIIIVal34Leu. Because of specificity and over a wide range sensitivity, the assay can also be used for determination of FXIII deficiency and monitoring of FXIII substitution therapy.
Subject(s)
Factor XIII/metabolism , Leucine/genetics , Valine/genetics , Amines/pharmacokinetics , Amino Acid Substitution , Ammonia/metabolism , Chromogenic Compounds/pharmacokinetics , Clinical Chemistry Tests/methods , Clinical Chemistry Tests/standards , Enzyme-Linked Immunosorbent Assay , Factor XIII/genetics , Factor XIII Deficiency/blood , Factor XIII Deficiency/congenital , Factor XIII Deficiency/genetics , Fibrinogen/metabolism , Humans , Kinetics , Point Mutation , Sensitivity and SpecificityABSTRACT
Apparent membrane permeation coefficients (Papp) of poorly water-soluble drugs such as indomethacin (IDM) and triamterene (TAT) were obtained by the chamber method using an isolated rat intestinal tissue after solubilization of the drugs by additives. For the additives, sodium deoxycholate (DOC), polyethylene glycol 600 (PEG 600), dimethylsulfoxide (DMSO), ethanol (EtOH), propylene glycol (PG), and rat bile were examined. Their concentrations were determined in ranges considered to be appropriate from the results of in vivo experiments and physiological findings. From the correspondence between this membrane permeability and in vivo bioavailability, we evaluated the validity of our in vitro experiment. On the basis of these evaluations, it was shown that 5% DMSO and 10% PEG 600, which did not affect the membrane integrity, were most appropriate additives for chamber experiments. Papp of IDM was greater than that of TAT, indicating that the order corresponded with that of in vivo bioavailability after oral administration of their PEG 600 solutions. Accordingly, it was concluded that Papp obtained by our in vitro system can be used to assess the in vivo bioavailability.
