Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.     

Liver-specific drug targeting by coupling to bile acids.

Bile acids are selectively taken up from portal blood into the liver by specific transport systems in the hepatocyte plasma membrane. Therefore, studies were performed to evaluate the potential of bile acids as shuttles to deliver drugs specifically to the liver. The alkylating cytostatic drug chlorambucil and the fluorescent prolyl-4-hydroxylase inhibitor 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-oxaproline-Gly were covalently linked via an amide bond to 7 alpha, 12 alpha,-dihydroxy-3 beta- (omega-aminoalkoxy)-5-beta-cholan-24-oic acid. The chlorambucil-bile acid conjugates S 2521, S 2539, S 2567, and S 2576 inhibited Na(+)-dependent [3H]taurocholate uptake in a concentration-dependent manner both into isolated rat hepatocytes and rabbit ileal brush border membrane vesicles, whereas the parent drug chlorambucil showed no significant inhibitory effect. The chlorambucil-bile acid conjugates were able to prevent photoaffinity labeling of bile acid binding proteins in rat hepatocytes by the photolabile [3H]7,7-azo derivative of taurocholic acid indicating their bile acid character. The chlorambucil-bile acid conjugate S 2577 was able to alkylate proteins demonstrating the drug character conserved in the hybrid-molecules. Liver perfusion experiments revealed a secretion profile of the chlorambucil-bile acid conjugate S 2576 into bile very similar to taurocholate compared to chlorambucil which is predominantly excreted by the kidney. 4-Nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-oxaproline-Gly- t-butylester (S 4404), a fluorescent peptide inhibitor of prolyl-4-hydroxylase, was not transported in intact form from portal blood into bile in contrast to its bile acid conjugate S 3744; about 25% of the peptide-bile acid conjugate S 3744 was secreted in intact form into bile within 40 min compared with less than 4% of the parent oxaprolylpeptide S 4404. In conclusion, these studies reveal that modified bile acid molecules can be used as "Trojan horses" to deliver a drug molecule specifically into the liver and the biliary system. This offers important pharmacological options for the development of liver-specific drugs.     

Influence of amino acid side-chain modification on the uptake system for beta-lactam antibiotics and dipeptides from rabbit small intestine

The influence of chemical modification of functional amino acid side-chains in proteins on the H(+)-dependent uptake system for orally active alpha-amino-beta-lactam antibiotics and small peptides was investigated in brush-border membrane vesicles from rabbit small intestine. Neither a modification of cysteine residues by HgCl2, NEM, DTNB or PHMB and of vicinal thiol groups by PAO nor a modification of disulfide bonds by DTT showed any inhibition on the uptake of cephalexin, a substrate of the intestinal peptide transporter. In contrast, the Na(+)-dependent uptake systems for D-glucose and L-alanine were greatly inhibited by the thiol-modifying agents. With reagents for hydroxyl groups, carboxyl groups or arginine the transport activity for beta-lactam antibiotics also remained unchanged, whereas the uptake of D-glucose and L-alanine was inhibited by the carboxyl specific reagent DCCD. A modification of tyrosine residues with N-acetylimidazole inhibited the peptide transport system and did not affect the uptake systems for D-glucose and L-alanine. The involvement of histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics and small peptides (Kramer, W. et al. (1988) Biochim. Biophys. Acta 943, 288-296) was further substantiated by photoaffinity labeling studies using a new photoreactive derivative of the orally active cephalosporin cephalexin, 3-[phenyl-4-3H]azidocephalexin, which still carries the alpha-amino group being essential for oral activity. 3-Azidocephalexin competitively inhibited the uptake of cephalexin into brush-border membrane vesicles. The photoaffinity labeling of the 127 kDa binding protein for beta-lactam antibiotics with this photoprobe was decreased by the presence of cephalexin, benzylpenicillin or dipeptides. A modification of histidine residues in brush-border membrane vesicles with DEP led to a decreased labeling of the putative peptide transporter of Mr 127,000 compared to controls. This indicates a decrease in the affinity of the peptide transporter for alpha-amino-beta-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics across the enterocyte brush-border membrane.     

Project portfolio selection under uncertainty with outsourcing opportunities

The paper presents a stochastic optimization model for project portfolio selection under uncertainty about the real efforts required for the execution of the work packages contained in the projects. As a subproblem, the assignment of the work to human resources and the distribution of work over time is addressed. The available workforce is assumed as multi-skilled. Required efforts are modeled as random variables. The recourse action for the case where the available capacities of the internal human resources do not suffice to cover the actual work times consists in delegating parts of the work to external human resources. The staffing-and-scheduling subproblem is solved by means of a Frank-Wolfe type algorithm. To solve the upper-level problem of project portfolio determination, a modification of the Variable Neighborhood Search (VNS) algorithm is applied. Experimental results for a benchmark of synthetically generated test instances and for an illustrative example from the E-Commerce Competence Center Austria are provided.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.     

GER1, a GDSL motif-encoding gene from rice is a novel early light- and jasmonate-induced gene

The reaction of the rice mutant HEBIBA differs from that of wild-type rice in that the mutant responds inversely to red light and is defective in the light-triggered biosynthesis of jasmonic acid (JA). Using the wild type and the HEBIBA mutant of rice in a differential display screen, we attempted to identify genes that act in or near the convergence point of light and JA signalling. We isolated specifically regulated DNA fragments from approximately 10 000 displayed bands, and identified a new early light- and JA-induced gene. This gene encodes an enzyme containing a GDSL motif, showing 38 % identity at the amino acid level to lipase Arab-1 in Arabidopsis thaliana. The GDSL CONTAINING ENZYME RICE 1 gene (GER1) is rapidly induced by both red (R) and far-red (FR) light and by JA. The results are discussed with respect to a possible role for GER1 as a negative regulator of coleoptile elongation in the context of recent findings on the impact of JA on light signalling.