Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption posthatch. Nutrient uptake is mediated by digestive enzymes and membrane bound transporter proteins. The objective of this study was to determine expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and small intestine of Leghorn and Cobb chickens derived from 22–30 wk (young) and 45–50 wk (old) breeder flocks. Genes examined included peptide transporter PepT1, amino acid transporters EAAT3, CAT1, and B0AT, monosaccharide transporters SGLT1 and GLUT5, and digestive enzymes APN and SI. Expression of these genes was measured in YSM at embryonic day (e) 11, 13, 15, 17, 19, 20, and 21 and small intestine at e15, e17, e19, e20, and e21. Absolute quantification real-time PCR assessed gene expression. PepT1, APN, and B0AT expression in YSM peaked between e15 and e17 and then decreased until e21, while expression increased over time in the small intestine. SGLT1 and EAAT3 expression increased over time in the small intestine and YSM. There was minimal gene expression of SI in the YSM, while the small intestine had high expression. GLUT5 and CAT1 expression decreased in the YSM, while peaking at e19 then decreasing in the small intestine. Breed and flock age affected expression levels in some genes. These results demonstrate that these genes show tissue- and development-specific patterns of expression and that the YSM expresses many digestive enzymes and nutrient transporters associated with the small intestine. / Master of Science

No description available.

<p>The aims of this thesis were to study the in vivo relevance of membrane transporters for intestinal absorption and the hepatic disposition of drugs in humans and preclinical models. Fexofenadine is a substrate for ABCB1 (P-glycoprotein) and members of the organic anion transporting polypeptide (OATP/SLCO) family. It is marginally metabolised in humans. </p><p>The influence of known inhibitors of ABCB1 and OATPs on the membrane transport and pharmacokinetics of fexofenadine was investigated in Caco-2 and porcine models and in humans. The permeability of fexofenadine remained low, even when significantly altered by the addition of an inhibitor. Using the Loc-I-Gut^® technique in vivo in humans, it was possible to see that the jejunal effective permeability of fexofenadine was unchanged when given with verapamil. However, the systemic exposure and apparent absorption rate of fexofenadine increased. This suggests that the first-pass liver extraction of fexofenadine was reduced by verapamil, probably through the inhibition of sinusoidal OATP-mediated and/or canalicular ABCB1-mediated secretion. The unchanged permeability can be explained by simultaneous inhibition of jejunal apical OATP-uptake and ABCB1-efflux, which would leave fexofenadine to be transported by passive trancellular diffusion. A Loc-I-Gut^® perfusion in the porcine model enabling blood sampling in the portal and hepatic veins and bile collection revealed increased jejunal permeability, but no subsequent verapamil-induced elevation in the systemic exposure of fexofenadine. This indicates a species-related difference in the localisation of and/or the substrate specificity of fexofenadine for the transporters involved. The absence of an effect on the first-pass liver extraction in the porcine model might be caused by the observed lower liver exposure of verapamil.</p><p>Finally, a novel intubation technique enabling dosing of fexofenadine in the jejunum, ileum and the colon showed that fexofenadine was absorbed less along the length the intestine in agreement with the properties of a low permeability drug.</p>

No description available.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), the most common life-shortening genetic disease among Caucasians. Although general features of the structure of CFTR have been predicted from homology models, the conformational changes that result in channel opening and closing have yet to be resolved. We created new closed- and open-state homology models of CFTR, and performed targeted molecular dynamics simulations of the conformational transitions in a channel opening event. The simulations predict a conformational wave that starts at the nucleotide binding domains and ends with the formation of an open conduction pathway. Experimentally confirmed changes in side-chain interactions are observed in all major domains of the protein. We also identified unique-to-CFTR substitutions that may have led to channel activity in CFTR. Molecular modeling and simulations are used to compare the effects of these substitutions against a canonical ABC transporter, and suggest that gain of channel function in CFTR may have risen from loss of ATPase function at its NBDs. The models and simulation add to our understanding of the mechanism of ATP-dependent gating in this disease-relevant ion channel.

ABC transporters consist of two transmembrane domains (TMDs) that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize transport via ATP binding and hydrolysis. Using site-directed mutagenesis, the role of highly conserved residues in the NBDs of Abcb1a was investigated. / In both NBDs of Abcb1a the A-loop aromatic residue is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine (618 in NBD1 and 1263 in NBD2) also appears to lie close to the ATP molecule. These four tyrosine residues were mutated to tryptophan and the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis was analyzed. Y618W and Y1263W enzymes had catalytic characteristics similar to wild-type (WT) Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including an &sim;10-fold increase in KM(ATP). Thus, Y397 and Y1040 play an important role in Abcb1a catalysis. / Since it was speculated that ABC transporters utilize a catalytic base to hydrolyse the beta-gamma phosphodiester bond of ATP, a search for that residue was undertaken. Six pairs of highly conserved acidic residues in the NBDs of Abcb1a were investigated. Removal of the charge in D558N and D1203N as well as in E552Q and E1197Q produced enzymes with severely impaired transport. These mutants were purified and characterized with respect to ATPase activity. Mutants D558N and D1203N retained some drug-stimulated ATPase activity and vanadate (Vi) trapping of 8-azido-[alpha32P]nucleotide confirmed slower basal and drug-stimulated hydrolysis. The E552Q and E1197Q mutants showed absence of ATPase activity but Vi trapping of 8-azido-[alpha 32P]nucleotide was observed, at a level similar to that of WT Abcb1a. Photolabelling by 8-azido-[alpha32P]nucleotide, in the presence or absence of drug, was also detected in the absence of Vi. The ATPase activity, binding affinity, and trapping properties of these glutamate residues were further analyzed. In addition to the E&rarr;Q mutants, the glutamates were individually mutated to D, N, and A. The double mutants E552Q/E1197Q, E552Q/K1072R, and K429R/E552Q were also analyzed. The results obtained suggest that 1) the length of the side-chain is important for the catalytic activity, whereas the charge is critical for full turnover to occur, 2) formation of the catalytic transition state does occur in the mutant site in the single-site mutants, suggesting that E552 and E1197 are not classical catalytic carboxylates, 3) steps after formation of the transition state are severely impaired in these mutant enzymes, 4) NBD1 and NBD2 are functionally asymmetric, and 5) the glutamates are involved both in NBD-NBD communication and transition-state formation through orientation of the linchpin residue.

The aims of this thesis were to study the in vivo relevance of membrane transporters for intestinal absorption and the hepatic disposition of drugs in humans and preclinical models. Fexofenadine is a substrate for ABCB1 (P-glycoprotein) and members of the organic anion transporting polypeptide (OATP/SLCO) family. It is marginally metabolised in humans. The influence of known inhibitors of ABCB1 and OATPs on the membrane transport and pharmacokinetics of fexofenadine was investigated in Caco-2 and porcine models and in humans. The permeability of fexofenadine remained low, even when significantly altered by the addition of an inhibitor. Using the Loc-I-Gut® technique in vivo in humans, it was possible to see that the jejunal effective permeability of fexofenadine was unchanged when given with verapamil. However, the systemic exposure and apparent absorption rate of fexofenadine increased. This suggests that the first-pass liver extraction of fexofenadine was reduced by verapamil, probably through the inhibition of sinusoidal OATP-mediated and/or canalicular ABCB1-mediated secretion. The unchanged permeability can be explained by simultaneous inhibition of jejunal apical OATP-uptake and ABCB1-efflux, which would leave fexofenadine to be transported by passive trancellular diffusion. A Loc-I-Gut® perfusion in the porcine model enabling blood sampling in the portal and hepatic veins and bile collection revealed increased jejunal permeability, but no subsequent verapamil-induced elevation in the systemic exposure of fexofenadine. This indicates a species-related difference in the localisation of and/or the substrate specificity of fexofenadine for the transporters involved. The absence of an effect on the first-pass liver extraction in the porcine model might be caused by the observed lower liver exposure of verapamil. Finally, a novel intubation technique enabling dosing of fexofenadine in the jejunum, ileum and the colon showed that fexofenadine was absorbed less along the length the intestine in agreement with the properties of a low permeability drug.

No description available.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anežka Lalinská Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of interactions of antiretroviral drug tenofovir and its prodrug tenofovir disoproxil fumarate with placental nucleoside transporters Tenofovir (TFV) in the form of ester prodrug tenofovir disoproxil fumarate (TDF) is an essential part of combination antiretroviral therapy. It is often used in the prevention of perinatal HIV transmission. However, precise mechanism(s) involved in transfer of TFV/TDF from mother to fetus are not described in detail. Since these drugs are nucleoside analogues, there is a possibility that the mechanisms of their transplacental passage might include nucleoside transporters (NTs), either equilibrative or concentrative (ENTs/CNTs). The aim of the diploma thesis was to investigate the role of placental NTs in membrane transfer of TFV and TDF. To address this issue, we performed in vitro accumulation in the BeWo cell line derived from placental choriocarcinoma. By evaluating experiments, we found out that both TFV and TDF might not be substrates of NTs, thus the role of these transporters in TFV/TDF placental pharmacokinetics was not confirmed. Therefore, the drug-drug interactions on NTs...

BACKGROUND:Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide.METHODS:To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS).RESULTS:3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters.CONCLUSIONS:The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-kappabeta and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.