Involvement of multidrug resistance-associated protein 2 (ABCC2/Mrp2) in biliary excretion of micafungin in rats

The drug transporter, multidrug resistance-associated protein 2 (ABCC2/Mrp2), is known to play important roles in excretion of various drugs. In the present study, we investigated whether Mrp2 is involved in the transport of micafungin, a newly developed antifungal agent. When Sprague-Dawley rats received an intravenous injection of micafungin (1 mg/kg) in combination with cyclosporine, the cyclosporine significantly delayed the disappearance of micafungin from plasma and decreased the systemic clearance and volume of distribution at steady-state of micafungin to 54% and 65% of the corresponding control values, respectively. When Sprague-Dawley rats received a constant-rate infusion of micafungin, cyclosporine significantly decreased the steady-state biliary clearance of micafungin (~80%). A significant decrease in the biliary clearance of micafungin (~60%) was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. The present findings at least suggest that Mrp2 is involved mainly in the hepatobiliary excretion of micafungin in rats.     

Allelic diversification at the <Emphasis Type="Italic">wx</Emphasis> locus in landraces of Asian rice

To examine continuous variation of amylose levels in Asian rice (Oryza sativa) landraces, the five putative alleles (Wx ^a , Wx ⁱⁿ , Wx ^b , Wx ^op , and wx) at the wx locus were investigated in near-isogenic lines (NILs). Apparent amylose levels ranged from 0.5 to 29.9% in the NILs, showing a positive relation with the levels of Wx gene product, granule-bound starch synthase (GBSS) as well as the enzymatic activity per milligram starch granule. Only opaque (Wx ^op ) accessions had an enzymatic activity per GBSS that was reduced to half the level of the others. Nucleotide sequences in the Wx gene were compared among 18 accessions harboring the five different alleles. Each of the Wx alleles had a unique replacement, frame-shift or splice donor site mutation, suggesting that these nucleotide changes could be reflected in phenotype alterations. A molecular phylogenetic tree constructed using the Wx gene indicated that ssp. japonica forms a distinct clade, whereas ssp. indica forms different clades together with the wild progenitor. Unexpectedly, the wx allele of 160 (indica from Taiwan) joined the japonica lineage; however, comparisons using linked genes for two Taiwanese accessions revealed that the wx gene was the product of gene flow from japonica to indica. Therefore, the japonica lineage frequently included Wx ⁱⁿ , Wx ^b and wx, while Wx ^a and Wx ^op were found in the other lineages, strongly suggesting that allelic diversification occurred after divergence of the two subspecies. The present results were discussed in relation to the maintenance of agronomically valuable genes in various landraces.     

Sequence dependence of fluorescence emission and quenching of doubly thiazole orange labeled DNA: effective design of a hybridization-sensitive probe

We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.     

Hypoxia induces expression of a GPI-anchorless splice variant of the prion protein

The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI(-) PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI(-) PrPSV expression in T98G cells.     

Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-alpha), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis

The role of TLRs and MyD88 in the maintenance of gut integrity in response to dextran sodium sulfate (DSS)-induced colitis was demonstrated recently and led to the conclusion that the innate immune response to luminal commensal flora provides necessary signals that facilitate epithelial repair and permits a return to homeostasis after colonic injury. In this report, we demonstrate that a deficit in a single neutrophil chemokine, CXCL1/KC, also results in a greatly exaggerated response to DSS. Mice with a targeted mutation in the gene that encodes this chemokine responded to 2.5% DSS in their drinking water with significant weight loss, bloody stools, and a complete loss of gut integrity in the proximal and distal colon, accompanied by a predominantly mononuclear infiltrate, with few detectable neutrophils. In contrast, CXCL1/KC(- /-) and wild-type C57BL/6J mice provided water showed no signs of inflammation and, at this concentration of DSS, wild-type mice showed only minimal histopathology, but significantly more infiltrating neutrophils. This finding implies that neutrophil infiltration induced by CXCL1/KC is an essential component of the intestinal response to inflammatory stimuli as well as the ability of the intestine to restore mucosal barrier integrity.     

Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation

Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.