Phase-contrast magnetic resonance (MR) imaging is a well-known but undervalued method of obtaining quantitative information on blood flow. Applications of this technique in cardiovascular MR imaging are expanding. According to the sequences available, phase-contrast measurement can be performed in a breath hold or during normal respiration. Prospective as well as retrospective gating techniques can be used. Common errors in phase-contrast imaging include mismatched encoding velocity, deviation of the imaging plane, inadequate temporal resolution, inadequate spatial resolution, accelerated flow and spatial misregistration, and phase offset errors. Flow measurements are most precise if the imaging plane is perpendicular to the vessel of interest and flow encoding is set to through-plane flow. The sequence should be repeated at least once, with a high encoding velocity used initially. If peak velocity has to be estimated, flow measurement is repeated with an adapted encoding velocity. The overall error of a phase-contrast flow measurement comprises errors during prescription as well as errors that occur during image analysis of the flow data. With phase-contrast imaging, the overall error in flow measurement can be reduced to less than 10%, an acceptable level of error for routine clinical use.

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y ؉ LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.

System L-type transport of large neutral amino acids is mediated by ubiquitous LAT1-4F2hc and epithelial LAT2-4F2hc. These heterodimers are thought to function as obligatory exchangers, but only in¯ux properties have been studied in some detail up until now. Here we measured their intracellular substrate selectivity, af®nity and exchange stoichiometry using the Xenopus oocyte expression system. Quanti®cation of amino acid in¯ux and ef¯ux by HPLC demonstrated an obligatory amino acid exchange with 1:1 stoichiometry. Strong, differential trans-stimulations of amino acid in¯ux by injected amino acids showed that the intracellular substrate availability limits the transport rate and that the ef¯ux selectivity range resembles that of in¯ux. Compared with high extracellular apparent af®nities, LAT1-and LAT2-4F2hc displayed much lower intracellular apparent af®nities (apparent K m in the millimolar range). Thus, the two system L amino acid transporters that are implicated in cell growth (LAT1-4F2hc) and transcellular transport (LAT2-4F2hc) are obligatory exchangers with relatively symmetrical substrate selectivities but strongly asymmetrical substrate af®nities such that the intracellular amino acid concentration controls their activity. Keywords: epithelial cell polarity/glycoproteinassociated amino acid transporter/LAT1-4F2hc/ LAT2-4F2hc IntroductionTwo heterodimeric transporters for large neutral amino acids that correspond to the Na + -independent system L have recently been identi®ed (Kanai et al., 1998;Mastroberardino et al., 1998;Pineda et al., 1999;Prasad et al., 1999;Rossier et al., 1999;Segawa et al., 1999;Rajan et al., 2000). These heterodimers contain catalytic subunits named LAT1 and LAT2, which belong to the family of glycoprotein-associated amino acid transporters (gpaATs) and are also called light chains (Verrey et al., , 2000. A disul®de bond covalently links these gpaATs to their associated glycoprotein 4F2hc/CD98.Functional experiments performed in expression systems suggest that the two L-type transporters function as exchangers (Mastroberardino et al., 1998;Pineda et al., 1999;Rossier et al., 1999). In the case of LAT2-4F2hc, some contradictory data have been published. Our laboratory and that of Palacin have shown that the ef¯ux of L-Phe and of L-Ile depend on the presence of an extracellular uptake substrate, as expected for an obligatory exchange (Pineda et al., 1999;Rossier et al., 1999). In contrast, an ef¯ux of L-Leu observed by Kanai and co-workers was interpreted as a facilitated diffusion (Segawa et al., 1999). Some functional differences between the two L-type transporters were reported, in particular the fact that LAT2-4F2hc has a broader selectivity range than LAT1-4F2hc in that it also mediates the uptake of smaller neutral amino acids.The tissue distribution and subcellular localization of the two L-type transporters suggest that they must play different roles. LAT1-4F2hc is found quite ubiquitously and is highly expressed in proliferating tissues, in particular also in tumors, sugges...

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na ⍣ -independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease-related protein (LAT1 or AmAT-L-lc) which for surface expression and function requires formation of disulfide-linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y ⍣ LAT1 from mouse and human which are~48% identical with LAT1 and thus belong to the same family of glycoprotein-associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na ⍣ -dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmacological fingerprints identify them as y ⍣ L-type transport systems. The mRNA encoding my ⍣ LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y ⍣ LAT1-4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in transepithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b0,+ type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b0,+AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b0,+amino acid transporter complex. We demonstrate its b0,+-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b0,+AT protein is the product of the gene defective in non-type I cystinuria.

This article examines the way in which microscopic tissue parameters affect the signal attenuation of diffusion-weighted MR experiments. The influence of transmembrane water flux on the signal decay is emphasized using the Kä rger equations, which are modified with respect to the cellular boundary restrictions for intra-and extracellular diffusion. This analytical approach is extensively compared to Monte-Carlo simulations for a tissue model consisting of two compartments. It is shown that diffusion-weighted MR methods provide a unique tool for estimation of the intracellular exchange time. Restrictions of applicability to in vivo data are examined. It is shown that the intracellular exchange time strongly depends on the size of a cell, leading to an apparent diffusion time dependence for in vivo data. Hence, an analytical model of a two-compartment system with an averaged exchange time is inadequate for the interpretation of signal curves measured in vivo over large ranges of b-values. During recent years, diffusion-weighted nuclear magnetic resonance methods (DW-NMR) have attracted attention in clinical diagnosis for the detection of brain lesions, especially of stroke in its early stage. The image contrast of this method is based on the differences in the apparent diffusion coefficients (ADC) of water in healthy and infarcted brain regions. The ADC reflects diffusion properties within tissue and depends on many physiological parameters such as volume fractions, extracellular tortuosity, intracellular restrictions, membrane permeability, and active processes across membranes, relaxation rates, or anisotropic morphology etc. Hence, the interpretation of diffusionweighted data is a complex multiparameter problem and it is crucial to extract the parameters specified above from the ADC in order to gain a better characterization of the physiological state of the tissue. Thus, much effort has been invested in the analytical description of diffusion effects on the NMR signal (see, for example, Refs. 1-4). However, any proposed mathematical model only provides an approximation with vague limits of applicability, in particular because many model input parameters, such as the intracellular diffusion coefficient, the tortuosity factor, or the exchange rates of water across cellular membranes are still not clearly known. Furthermore, their effect on the signal attenuation depends on the experimental parameters, such as diffusion time or gradient strength. Therefore, analytical models have often been compared to numerical simulations (2,3,5). Many experiments performed in vitro and in vivo showed that the diffusion attenuation is not monoexponential when measured over a wide range of b-values (3,6 -14). This behavior presumably reflects tissue compartmentalization and may provide structural information. Hence, deviations from monoexponentiality can be used to separate the influence of tissue parameters on the attenuation and may therefore be exploited when testing analytical models. This is the approach set forth in this a...

The intracellular delivery of active biomacromolecules from endosomes into the cytoplasm generally requires a membrane-disrupting agent. Since endosomes have a slightly acidic pH, anionic carboxylated polymers could be potentially useful for this purpose since they can destabilize membrane bilayers by pH-triggered conformational change. In this study, five different pH-sensitive methacrylic acid (MAA) copolymers were characterized with respect to their physicochemical and membrane lytic properties as a function of pH. pH-dependent conformational changes were studied in aqueous solution by turbidimetry and spectrofluorimetry. The hydrophobic domains that formed upon a decrease in pH were found to be dependent on copolymer's composition. Hemolysis and cytotoxicity assays demonstrated that the presence of the hydrophobic ethyl acrylate monomer and/or sufficient protonation of the carboxylic acid groups were important parameters for efficient membrane destabilization. Excessive copolymer hydrophobicity was not associated with membrane destabilization, but resulted in high macrophage cytotoxicity. Overall, this study gave more insights into the structure-activity relationship of MAA copolymers with membrane bilayers. Gaining knowledge of modulation of the physicochemical properties of copolymers and the optimization of copolymer-lipid interactions may lead to the elaboration of much more efficient drug delivery systems.

Enzyme recycling is essential for the development of large-scale enzyme-catalyzed biotransformations. Recycling is most convenient using enzymes immobilized on solid supports. Although immobilization on solid supports has been pursued since the 1950s, there are no general rules for selecting the best support for a given application. The commercial products EUPERGIT C and EUPERGIT C 250 L have been used for a wide variety of different enzymes and reactions. The present review draws up a comprehensive application profile of both EUPERGIT carriers. The reader gets (a) examples of biotransformations using oxidoreductases, transferases, hydrolases and lyases immobilized on EUPERGIT; (b) key data of the biotransformations, i.e., scale, yield, purity, and enantiomeric excess; (c) efficiency of the immobilization (% immobilized activity); (d) where appropriate, operational stability of the immobilized enzyme preparations, that is, number of cycles, residual activity; (e) specific advantages of the immobilized enzyme over the free enzyme apart from enzyme recycling, for example, improved stability and selectivity. Thus, the present review can serve as a guideline when selecting a resin for enzyme immobilization. Literature published between 1985 and 2000 is covered.