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Carsten Kneuer

Publications and source records attributed to Carsten Kneuer.

14 recordsLinked to original sources
PubMed2006

Selectins--potential pharmacological targets?

Recent advances in our (patho)physiological understanding have underpinned the frequent involvement of the protein family of selectins in the progression of serious illnesses, including cancer and cancer metastasis, and immunological diseases, such as asthma, allergy and autoimmune reactions. Moreover, selectins seem to have a role in post-ischemic damage and during transplant failures (e.g. in graft-versus-host disease). Although the interplay between selectins and their counter-receptors and ligands is not always primarily involved in the development of these pathological conditions, selectins have been investigated as potential therapeutic targets for therapeutic intervention. This review focuses on the latest trends and developments in anti-selectin antibodies, anti-selectin receptor antibodies, recombinant selectin counter-receptors, low molecular weight selectin antagonists (glycomimetics), induction of selectin tolerance and selectin-targeted imaging agents.

Animals↗
PubMed2006

Alternative transcripts of rat slc19a1: defective sorting and inefficient expression of alternative splicing products--an addendum.

Recently, we have reported about the existence and cloning of alternative transcripts of the solute carrier 19a1 in rat liver and kidney, and have explained their origin from tissue specific promoters and by alternative splicing [DNA Seq. 2005, 16(1), 1-6]. The variant open reading frames (ORFs) of these transcripts were now expressed as fusion proteins with an N-terminal GFP. Transfection of HPCT-1E3 hepatocytoma cells as well as the MDCK kidney epithelial cell line with the GFP-fusion of the major splicing form containing 12 transmembrane domains (TMD) resulted in a strong fluorescence at the plasma membrane, which was stable over at least 3 days. Expression of GFP alone gave a comparable overall fluorescence intensity, although, staining was distributed evenly throughout the cells. Fusions of GFP with the shorter ORFs of all alternative slc19a1 transcripts containing 6-7 predicted TMD were expressed less efficiently and not sorted to the plasma membrane. Instead, these proteins accumulated in intracellular granules, and were, apparently, degraded. Hence, it is unlikely that these minor splicing forms are directly involved in solute transport across the plasma membrane.

Alternative Splicing↗
PubMed2005

Rat reduced-folate carrier-1 is localized basolaterally in MDCK kidney epithelial cells and contributes to the secretory transport of methotrexate and fluoresceinated methotrexate.

The reduced-folate carrier (Rfc-1), previously also called methotrexate carrier-1 (MTX-1), was recently identified as accounting for approximately 30% of the methotrexate (Mtx) uptake into rat kidney slices. The localization of the carrier and its contribution to secretory or reabsorptive flux of the drug was therefore evaluated in polarized epithelial layers of Madin Darby canine kidney (MDCK) cells. Confocal laser scanning microscopy revealed that the HA-epitope-tagged protein was sorted to the basolateral side. In flux assays, the basolateral-to-apical transport of fluoresceinated methotrexate (FMTX) was two-fold higher than in the apical-to-basolateral direction across rat Rfc-1 transfected, but not mock-transfected, monolayers. The same observation was made for unlabeled Mtx. This secretory transport of FMTX was inhibited by an excess of 1 mM Mtx and was saturable and temperature-dependent. No differences in directional flux were observed for the pure fluorescein label. Removal of sodium resulted in a marked decrease of directional FMTX flux. The pH profile of the active transport component showed a trough around 6.5 and a maximum at acidic pH, as reported for uptake into Rfc-1-expressing cells. Thus, rat Rfc-1 is sorted to the basolateral side in polarized MDCK epithelial cells and mediates the secretion of Mtx, probably in co-operation with efflux proteins, such as multidrug resistance associated proteins, which are also expressed in these cells.

Animals↗
PubMed2005

Endogenous expression of liver-specific drug transporters for organic anions in the rat hepatocytoma fusion cell line HPCT-1E3.

HPCT-1E3 cells, a fusion cell line between primary rat hepatocytes and Fao Reuber hepatoma cells H35, are immortalized hybrid cells with many phenotypic properties of liver parenchyma including phase I and II metabolism and bile acid secretion. Selective elimination of endogenous compounds and drugs by the liver involves transport proteins that complementarily mediate uptake and efflux in co-operation with metabolism, but the study of this function is limited by the unavailability of an integrated in vitro model. Therefore, we investigated the expression of some important liver-specific import and export carrier proteins for organic anions in this cell line. RT-PCR analysis indicated gene expression of Oat2, Oatplal, Oatpla4, Oatplb2, Rfc-1/MTX-1, FOLR, Mrp1-6, mdr1, and Lrp. Uptake and efflux as well as inhibition studies confirmed the functional activity of Oat, Oatp, Rfc-1, Mrp, and Mdr carriers. In conclusion, the hepatocyte-like HPCT-1E3 cell line shows endogenous expression of all liver-specific carrier proteins for organic anions and may hence represent a valuable in vitro model for the study of transport phenomena and their regulation in hepatocytes.

Animals↗
PubMed2005

Alternative transcripts of rat slc19a1: cloning, genomic organisation, tissue specific promoters and alternative splicing.

Recently, the rat genome project revealed the genomic sequence of slc19a1, coding for the methotrexate carrier-1, identical to the reduced folate carrier-1 of humans, on rat chromosome 20. At the same time, we have cloned and analysed the complete or partial cDNAs of now at least six different transcripts from rat liver and kidneys. Alignment with the genomic sequence revealed seven exons. The first two non-coding exons, exon I and Ia were used alternatively in kidneys and liver, respectively, suggesting usage of alternative promoters. Three minor mRNA forms resulted from absent splicing of intron III, a shortened exon III (exon IIIa), and a shortened exon IV (exon IVa). The minor transcripts were predicted to result in translation products with 7 or 6 instead of 12 transmembrane domains (TMDs) and a peptide mass of 38, 39 and 40 kDa instead of 58 kDa.

Alternative Splicing↗
PubMed2004

Transfection with different colloidal systems: comparison of solid lipid nanoparticles and liposomes.

Cationic solid lipid nanoparticles (SLN) for gene transfer are formulated using the same cationic lipids as for liposomal transfection agents. To investigate the differences and similarities in structure and performance between SLN and liposomes, a SLN preparation (S1), its counterpart formulation without matrix lipid (L1), a commercially available liposomal preparation (DLTR)--all based on the cationic lipid DOTAP--and a liposomal formulation that additionally contained the helper lipid dioleoylphosphatidylethanolamine (DOPE) (Escort) were compared. Photon correlation spectroscopy (PCS) showed that the SLN were smaller in diameter than the corresponding liposomes (88 vs. 148 nm) and atomic force microscopy (AFM) supported the expected structural differences. Desoxy ribonuclein acid (DNA) binding differed only marginally. Surprisingly, reporter gene expression was comparable between all DOTAP based formulations (S1, L1, DLTR), surpassed only by the DOPE containing liposomes (Escort). In conclusion, cationic lipid composition seems to be more dominant for in vitro transfection performance than the kind of colloidal structure it is arranged in. Hence, cationic SLN extend the range of highly potent non-viral transfection agents by one with favourable and distinct technological properties. Further SLN optimisation should be facilitated by the accumulated knowledge about cationic lipids in liposomal formulations.

Animals↗
PubMed2004

The H(+)-dependent reduced folate carrier 1 of humans and the sodium-dependent methotrexate carrier-1 of the rat are orthologs.

Previously, two different carrier systems for uptake of reduced folates and the antifolate methotrexate (Mtx) were described: the pH-dependent folate sensitive reduced folate carrier 1 (RFC1) from human, hamster and mouse and a sodium-dependent and folate insensitive Mtx carrier-1 (MTX-1) from rat. It was found that all critical residues of the homologous amino acid sequence were identical. RFC1- as well as MTX-1-mediated uptake of a marker substrate into suitable human and rat cell lines increased with proton concentration, was sodium-dependent at neutral pH, and inhibited by folate at acidic pH. It is concluded that RFC1 and MTX-1 are orthologs.

Amino Acid Sequence↗
PubMed2004

Expression of functional beta2-adrenergic receptors in the lung epithelial cell lines 16HBE14o(-), Calu-3 and A549.

Adrenergic drugs acting through the beta(2)-adrenoceptor (beta(2)-AR) adenylate cyclase (AC) signal transduction system elicit a variety of responses within the mammalian airway epithelium; however, its composition of multiple phenotypically differentiated cell types complicates the understanding of the regulation cascades within this tissue. The present study evaluates beta(2)-AR mRNA level, number, subtype and the cyclic adenosine-3',5'-monophosphate (cyclic AMP) response to isoproterenol (iso) in the human airway epithelial cell lines 16HBE14o(-), Calu-3 and A549, using reverse transcriptase polymerase chain reaction (RT-PCR), radioligand binding studies, [(3)H]-radioimmunoassay and immunocytochemical staining. After 4-5 days in culture, all three cell types produced beta(2)-AR mRNA and protein at a magnitude of gene expression levels Calu-3>or=16HBE14o(-)>A549, whereas control cells Cos-1 and Caco-2 were negative. The beta(2)-AR adenylate cyclase system was highly expressed and functional in the human airway epithelial cells Calu-3 and 16HBE14o(-). The mean beta(2)-AR density (B(max)), equilibrium dissociation constant (K(D)), and the percentage of beta-AR subtypes assessed by radioligand binding were approximately 9908+/-1127 and 6423+/-895 binding sites/cell, 32+/-2.7 pM and 25+/-1.1 pM, and approximately 100% in Calu-3 and 16HBE14o(-)cells, respectively. However, in the alveolar cell type A549 the cell surface beta(2)-AR was virtually undetectable by (-)-[(125)I]-iodocyanopindolol (ICYP) binding. Stimulation of cultured cells with (-)-isoproterenol enhanced the basal cyclic AMP accumulation only in Calu-3 and 16HBE14o(-) cells, which was blocked by the beta(2)-selective antagonist ICI 118,551, but not by the beta(1)-selective antagonist CGP 20712A, confirming functional coupling of the beta(2)-AR to adenylate cyclase in these cells. Immunocytochemical staining localised the receptor on the cell membrane and the cytoplasm in Calu-3 and 16HBE14o(-) cells, while it was confined to the cytoplasm only in A549 cells. In conclusion, the beta(2)-AR expression and its functional coupling to adenylyl cyclase was very high in the human airway epithelial cells Calu-3 and 16HBE14o(-), but not in A549, suggesting that the cell lines Calu-3 and 16HBE14o(-) present suitable models to study function and regulation of the beta-adrenoceptor signalling in the respiratory system.

Adenylyl Cyclases↗
PubMed2004

Selectins-an emerging target for drug delivery.

Selectins are multifunctional adhesion molecules that mediate the initial interactions between circulating leukocytes and cells of the endothelium. First identified over a decade ago, selectins have provided insight into areas as diverse as normal lymphocyte homing, leukocyte recruitment during inflammatory responses, carbohydrate ligand biosynthesis and adhesion-mediated signalling. Of late, selectins were introduced as targets for drug delivery in the development of new anti-inflammatory therapeutics and in anti-cancer therapy. This review will examine the selectins and their ligands with a focus on recent findings on their role in physiology and pathophysiology as well as the emerging role of selectins as targets in controlled drug delivery.

Animals↗
PubMed2003

Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization.

Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5'-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a K(m) of 41 microM and a V(max) of 337 pmol.mg protein(-1).min(-1) was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model.

Animals↗
PubMed2003

16HBE14o- human bronchial epithelial cell layers express P-glycoprotein, lung resistance-related protein, and caveolin-1.

PURPOSE: To study the expression of P-glycoprotein (P-gp), lung resistance-related protein (LRP), and caveolin-1 (cav-1) in the human bronchial epithelial cell line 16HBE14o-. METHODS: The presence of P-gp, LRP, and cav-1 in 16HBE14o- cell layers was evaluated using immunocytochemical staining and visualization with confocal laser scanning microscopy (CLSM). Functionality of P-gp was determined by bidirectional transport of rhodamine-123 with and without a P-gp inhibitor, verapamil. Caveolae were visualized using transmission electron microscopy (TEM). Flux of fluorescein-Na was also studied as a paracellular transport marker. RESULTS: Immunocytochemical staining showed expression of P-gp localized at the apical membrane of 16HBE14o- cell layers. The flux of rhodamine 123 across cell layers exhibited a greater Papp value for the secretory (i.e., basolateral-to-apical) direction. This asymmetry disappeared in the presence of verapamil. CLSM provided evidence for the expression of LRP and cav-1. TEM further showed typically shaped caveolae at the apical and basolateral membranes. CONCLUSION: Cell layers of 16HBE14o- express drug transport systems that are also present in the human bronchus in vivo, indicating that the 16HBE14o- cell line may be a suitable candidate for an in vitro model for mechanistic studies of drug transport processes involved in the smaller airways.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies.

Air-interfaced culture (AIC) versus liquid-covered culture (LCC) conditions are known to have different effects on the differentiated phenotype of several cell types, including lung epithelial cells. We report the influence of culture conditions such as apical medium volume on the development of intercellular junctions in the human epithelial cell line 16HBE14o-. Immunofluorescence staining of the tight-junctional protein, ZO-1, has revealed its presence in cells grown in both AIC and LCC. However, only LCC-grown cells exhibit protein ZO-1 localized as a zonula-occludens-like regular belt connecting neighboring cells. The presence of typical tight junctions has been confirmed by electron microscopy. Immunostaining for occludin, claudin-1, connexin43, and E-cadherin has demonstrated intercellular junction structures only in the cells in LCC. These morphological findings have been paralleled by higher transepithelial electrical resistance values and similar fluxes of the hydrophilic permeability marker, fluorescein-Na, under LCC compared with AIC conditions. We conclude that the formation of functional 16HBE14o- cell layers requires the presence of an apical fluid volume, in contrast to other culture conditions for airway epithelial cells.

Actins↗
PubMedDate not supplied

The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes: a comparative study.

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class of vector. To investigate the general validity of these criteria, we studied the efficacy of a variety of DNA delivery vehicles including liposomes (DOTAP, SAINT2) with and without helper lipid (DOPE), the polymer polyethyleneimine (PEI), and cationic nanoparticles (Si26H, PLGA/chitosan) in a comparative manner. Sizes of the DNA complexes varied between 100 and 500 nm for PEI polyplexes and DOTAP/DOPE lipoplexes, respectively. The zeta potential was positive for PEI, Si26H, and DOTAP based complexes, while it was neutral for SAINT2-DNA complexes and negative for PLGA/chitosan-DNA complexes. The latter finding was elucidated by AFM, showing a layer of DNA adsorbed onto the nanoparticles. Transfection activity was negligible for PLGA/chitosan nanospheres, moderate for Si26H nanospheres and high for all other complexes, PEI being the most active carrier. The liposomal preparations were of low (DOTAP) or moderate (SAINT2) stability in serum, resulting in a pronounced reduction of gene expression, which was partially restored by the addition of chloroquine. In conclusion, transfection efficiency (i) seems to require a positive or neutral zeta potential, (ii) is depending on size, e.g., is higher for smaller particles, and (iii) requires a vector that is stable in serum.

Animals↗