Labeo rosae (Cypriniformes: Cyprinidae) in the Congo basin: a relict distribution or a historical introduction?

Labeo rosae, a species with a native range in Southern Africa, was discovered in the Congo basin by re-identification of two museum specimens previously identified as Labeo mesops. The occurrence of this species in the upper Congo implies a range extension of the species of more than 1000 km. Although the species' distribution is mirrored by that of some other Cypriniformes, its occurrence in the Congo might be due to introduction by humans.

Dose-dependent food allergy induction against ovalbumin under acid-suppression: a murine food allergy model.

BACKGROUND: Animal models are essential for analyzing the allergenic potential of food proteins and for investigating mechanisms underlying food allergy. Based on previous studies revealing acid-suppression medication as risk factor for food allergy induction, we aimed to establish a mouse model mimicking the natural route of sensitization in patients. METHODS: The effect of acid-suppressing medication on murine gastric pH was assessed by intragastric pH measurements after two injections of a proton pump inhibitor (PPI). To investigate dose-dependency, mice were fed different concentrations of ovalbumin (OVA; 0.2, 0.5, 1.0, 2.5 or 5.0mg) either with or without anti-ulcer medication. Additionally, different routes of exposure (i.p. vs. oral) were compared in a second immunization experiment. Sera were screened for OVA-specific antibody titers (IgG1, IgG2a and IgE) in ELISA and RBL assay. Clinical reactivity was evaluated by measuring rectal temperature after oral challenge and by type I skin tests. RESULTS: Two intravenous injections of PPI significantly elevated the gastric pH from 2.97 to 5.3. Only oral immunization with 0.2mg OVA under anti-acid medication rendered elevated IgG1, IgG2a and IgE titers compared to all other concentrations. Protein feeding alone altered antibody titers only marginally. Even though also i.p. immunizations induced high levels of specific IgE, only oral immunizations under anti-acids induced anaphylactic reactions evidenced by a significant decrease of body temperature. CONCLUSION: Only low-dosage ovalbumin feedings under anti-acid medication resulted in IgE mediated food allergy. Based on this knowledge we have established a suitable food allergy model for further investigations of food adverse reactions.

Electrogenicity of hepatocellular fatty acid uptake.

Sensitivity of cellular fatty acids uptake to the membrane potential difference is still a matter of controversy. For direct evaluation of potential sensitivity the effect of changing membrane potential on uptake of a fluorescent long chain fatty acid derivative, 12-NBD-stearate, in isolated rat hepatocytes, was examined. Changes in membrane potential were achieved by patch clamp procedures. Fatty acid influx was simultaneously determined by recording of cell fluorescence. Hyperpolarization from -30 to -70 mV accelerated fatty acid influx whereas depolarization to +50 mV reduced uptake. After obtaining equilibrium hyperpolarization increased cell fluorescence, whereas depolarization pushed NBD-stearate out of cells. Potential sensitivity of uptake was dependent on the fatty acid concentrations in the medium with most prominent effects at low unbound concentrations. These data show that, at low fatty acid concentrations, uptake is, in part, driven by an intracellular negative electric membrane potential.

Hot spices influence permeability of human intestinal epithelial monolayers.

Indirect evidence suggests that hot spices may interact with epithelial cells of the gastrointestinal tract to modulate their transport properties. Using HCT-8 cells, a cell line from a human ileocoecal carcinoma, we studied the effects of spices on transepithelial electrical resistance (TER), permeability for fluorescein isothiocyanate (FITC)-labeled dextrans with graded molecular weight, and morphological alterations of tight junctions by immunofluorescence using an anti-ZO-1 antibody, a marker for tight junction integrity. Two different reactivity patterns were observed: paprika and cayenne pepper significantly decreased the TER and increased permeability for 10-, 20- and 40-kDa dextrans but not for -70 kDa dextrans. Simultaneously, tight junctions exhibited a discontinuous pattern. Applying extracts from black or green pepper, bay leaf or nutmeg increased the TER and macromolecular permeability remained low. Immunofluorescence ZO-1 staining was preserved. In accordance with the above findings, capsaicin transiently reduced resistance and piperine increased resistance, making them candidates for causing the effects seen with crude spice extracts. The observation that Solanaceae spices (paprika, cayenne pepper) increase permeability for ions and macromolecules might be of pathophysiological importance, particularly with respect to food allergy and intolerance.

Vitamin D increases tight-junction conductance and paracellular Ca2+ transport in Caco-2 cell cultures.

We investigated the effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] on paracellular intestinal Ca2+ absorption by determination of transepithelial electric resistance (TEER), as a measure of tight-junction ion permeability and bidirectional transepithelial 45Ca2+ fluxes in confluent Caco-2 cell cultures. The rise of TEER to steady-state levels of approximately 2,000 omega.cm2 was significantly attenuated by 1,25(OH)2D3 (by up to 50%) in a dose-dependent fashion between 10(-11) and 10(-8) M. Synthetic analogs of 1,25(OH)2D3, namely, 1 alpha,25-dihydroxy-16-ene,23-yne-vitamin D3 and 1 alpha,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3, exhibited similar biopotency, whereas their genomically inactive 1-deoxy congeners were only marginally effective. Enhancement of transepithelial conductance of Caco-2 cell monolayers by vitamin D was accompanied by a significant increase in bidirectional transepithelial 45Ca2+ fluxes. Although 1,25(OH)2D3 also induced cellular 45Ca2+ uptake from the apical aspect of Caco-2 cell layers and upregulated the expression of calbindin-9kDa mRNA, no significant contribution of the Ca(2+)-adenosinetriphosphatase-mediated transcellular pathway to transepithelial Ca2+ transport could be detected. Therefore stimulation of Ca2+ fluxes across confluent Caco-2 cells very likely results from a genomic effect of vitamin D sterols on assembly and permeability of tight-junctional complexes.

Modulation of liver canalicular transport processes by the tyrosine-kinase inhibitor genistein: implications of genistein metabolism in the rat.

Rat liver cells express the multispecific organic anion transporter (cmoat, cmrp, mrp2) and P-glycoprotein (Pgp) in their canalicular membranes, proteins that are homologous to the multidrug-resistance related protein (MRP) and multidrug resistance (MDR) gene products in multidrug resistant tumor cells. We tested whether genistein, a modulator of drug resistance in tumor cells, affects biliary secretion of substrates of canalicular multispecific organic anion transporter (cmoat) (glucuronides of bilirubin and rhodamine, glutathione conjugate of bromsulphthalein) and of P-glycoprotein (Pgp) (rhodamine), respectively. Using the isolated perfused rat liver of control Wistar rats (TR+) and of a mutant strain (TR-) that expresses Pgp but not cmoat, we show that genistein effectively inhibits the secretion of anionic substrates of cmoat in Wistar rats but stimulates secretion of cationic rhodamine in TR- rats. Genistein is subject to glucuronidation and sulfatation and secretion of genistein and its metabolites stimulates bile flow in Wistar rats, but secretion is nearly absent in TR- rats. Because genistein and its metabolites are substrates for cmoat, inhibition of anion secretion by genistein is partially explained by competition for this transporter. Genistein is also a substrate of uridindiphosphate (UDP)-glucuronyltransferase isoenzyme(s). Inhibition of glucuronidation reduces the availability of bilirubin and rhodamine glucuronates for transport via cmoat, but unconjugated cationic rhodamine becomes available for transport via Pgp at an increased cellular concentration. Daidzein, a genistein analogue with no effect on protein tyrosine kinase (PTK) shows Similar effects on secretion of organic anions and cations supporting the conclusion that genistein affects transport in liver mainly through competition with other substrates at the sites of glucuronidation and transport via cmoat.

High precision measurement of electrical resistance across endothelial cell monolayers.

Effects of vasoactive agonists on endothelial permeability was assessed by measurement of transendothelial electrical resistance (TEER) of human umbilical vein endothelial cells (HUVECs) grown on porous polycarbonate supports. Because of the low values of TEER obtained in this preparation (< 5 omega cm2) a design of an Ussing type recording chamber was chosen that provided for a homogeneous electric field across the monolayer and for proper correction of series resistances. Precision current pulses and appropriate rates of sampling and averaging of the voltage signal allowed for measurement of < 0.1 omega resistance changes of the endothelium on top of a 21 omega series resistance of the support and bathing fluid layers. Histamine (10 microM) and thrombin (10 U/ml) induced an abrupt and substantial decrease of TEER, bradykinin (1 microM) was less effective, PAF (380 nM) and LTC4 (1 microM) had no effect. TEER was also reduced by the calcium ionophore A-23187 (10 microM). The technique allows for measurements of TEER in low resistance monolayer cultures with high precision and time resolution. The results obtained extend previous observations in providing quantitative data on the increase of permeability of HUVECs in response to vasoactive agonists.

Bile canalicular cationic dye secretion as a model for P-glycoprotein mediated transport.

This study explores properties of P-glycoprotein dependent membrane transport in rat liver with the use of acridine orange as the substrate. We studied the biliary secretion of the dye, its binding to canalicular membrane P-glycoprotein, and effects of the inhibitor cyclosporin A: acridine orange is excreted into bile together with less hydrophobic and glucuronidated metabolites. Cyclosporin A inhibited both the secretion of acridine orange and of its metabolites. In TR- animals, a rat strain that is deficient of the canalicular multi-specific organic anion transport system, non-metabolized acridine orange is the predominant species in bile and its secretion is also inhibited by cyclosporin A. Binding of acridine orange to liver P-glycoprotein was analyzed by photoaffinity labeling with azidopine, a substrate of P-glycoprotein dependent transport in multi-drug resistant tumor cells. Labeling of the immunoprecipitated P-glycoprotein was inhibited by acridine orange, verapamil, and by cyclosporin A. The results show that biliary secretion of acridine orange is highly analogous to P-glycoprotein mediated membrane drug transport in tumor cells that exhibit multi-drug resistance.

Inhibition of rhodamine 123 secretion by cyclosporin A as a model of P-glycoprotein mediated transport in liver.

The interaction between P-glycoprotein modulators and P-glycoprotein mediated transport was investigated using rhodamine 123 in the isolated perfused rat liver of a mutant (TR-) rat strain. TR- rats, deficient in the canalicular multispecific anion transport system, are unable to extrude organic anions (glucuronides) and therefore excrete solely unconjugated rhodamine 123 via P-glycoprotein. Cyclosporin A, a modulator of multidrug resistance in tumor cells, inhibited the biliary secretion of rhodamine 123 dose dependently in a non-competitive manner. Both cyclosporin A and rhodamine inhibited photoaffinity labeling of immunoprecipitated P-glycoprotein with azidopine, indicating binding to hepatic P-glycoprotein. Our results indicate that monitoring the biliary rhodamine 123 secretion in the isolated perfused liver of TR- rats offers a new system for testing modulators of P-glycoprotein like cyclosporin A.