Monitoring of urinary acrolein concentration in patients receiving cyclophosphamide and ifosphamide

Acrolein, the metabolite of cyclophosphamide and ifosphamide, irritates mucous membranes and is considered pathogenetically important in hemorrhagic cystitis. Increasing fluid intake or administering sodium 2-mercaptoethanesulfonate (mesna), a thiol compound, can reduce the risk of this complication. We measured urinary acrolein concentrations using headspace-solid-phase microextraction gas chromatography and mass spectrometry (headspace-SPME-GC-MS) in 19 patients receiving cyclophosphamide and ifosphamide (36 occasions). Peak acrolein concentrations occurred at 1-12h (mean +/- S.D., 5.0+/-2.7) after starting therapy, ranging from 0.3 to 406.8 nM (39.7+/-76.7), with varying patterns over time. Maintaining high urine volume was important for preventing increases in urinary acrolein concentration, as urinary acrolein concentration tended to rise as urine volume decreased. Urinalysis detected occult blood in three cases, but the patients had no clinical symptoms of hemorrhagic cystitis. In clinical trials involving cyclophosphamide and ifosphamide, monitoring of urinary acrolein concentration could indicate when to take heightened preventive measures against hemorrhagic cystitis.     

Efficient asymmetric syntheses, determination of absolute configurations and biological activities of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine as a novel potent dopamine uptake inhibitor in the central nervous system

An efficient asymmetric synthesis of the chiral N-(3-chloro-2-hydroxypropyl)anilines (2a and 2b) was achieved through the regioselective ring-opening reaction of chiral epichlorohydrin with aniline. This was applied to an asymmetric synthesis of the enantiomers of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1 as a novel potent dopamine uptake inhibitor. Both enantiomers as trihydrochlorides, 4a.3HCl and 4b.3HCl, could be synthesized in good total yields and optical purities of 100% ee in three steps synthesis, respectively. The absolute configurations of 4a.3HCl and 4b.3HCl were determined using the modified Mosher's method with the related compounds, the intermediates (2a and 2b) and the free bases (4a and 4b). The analytical results indicated that 4a.3HCl and 4b.3HCl have the (S)- and (R)-configuration, respectively, and a series of reactions to provide them proceeded without the apparent influence on the stereochemistry at the chiral centers. In in vitro pharmacological evaluations, 4a.3HCl and 4b.3HCl showed potent dopamine transporter binding affinities, high dopamine, moderate serotonin, and weak norepinephrine uptake inhibitory activities, and 4a.3HCl exhibited a more potent and selective dopamine uptake inhibition over the serotonin or norepinephrine uptake inhibition as compared with 4b.3HCl. An ex vivo evaluation revealed that the oral administrations of both enantiomers at a dose of 30 mg/kg in rats displayed apparent dopamine uptake inhibitory activities and 4a.3HCl had a stronger tendency to inhibit dopamine uptake compared with 4b.3HCl.     

Synthesis and fungicidal activity of enantiomerically pure (R)- and (S)-silicon-containing azole fungicides

Enantiomerically pure (R)- and (S)-1-(1H-1,2,4-triazol-1-yl)-2-(4-fluorophenyl)-3-trimethylsilylpropan-2-ol 1 were prepared via an enantioselective Grignard reaction. The absolute stereochemistry of 1 was determined by X-ray analysis. In a comparison of in vitro antifungal activities of the enantiomers, the (-)-enantiomer with the R-absolute configuration was far more potent than the (+)-enantiomer.     

Composition and structure of the centromeric region of rice chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.     

Synthesis and structure-activity relationship for new series of 4-phenoxyquinoline derivatives as specific inhibitors of platelet-derived growth factor receptor tyrosine kinase

We discovered a new series of 4-phenoxyquinoline derivatives as potent and selective inhibitors of the platelet-derived growth factor receptor (PDGFr) tyrosine kinase. We researched the highly potent and selective inhibitors on the basis of both PDGFr and epidermal growth factor receptor (EGFr) inhibitory activity. First, we found a compound, Ki6783 (1), which inhibited PDGFr autophosphorylation at 0.13 microM, but it did not inhibit EGFr autophosphorylation at 100 microM. After extensive explorations, we found the two desired compounds, Ki6896 (2) and Ki6945 (3), which are substituted by benzoyl and benzamide at the 4-position of the phenoxy group on 4-phenoxyquinoline, respectively. These inhibitory activities were 0.31 and 0.050 microM, respectively, but neither of them inhibited EGFr autophosphorylation at 100 microM. We further investigated the profile of both compounds toward various tyrosine and serine/threonine kinases. The three compounds specifically inhibited PDGFr rather than the other kinases.     

Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Intracellular delivery of glutathione S-transferase into mammalian cells

Protein transduction domains (PTDs) derived from human immunodeficiency virus Tat protein and herpes simplex virus VP22 protein are useful for the delivery of non-membrane-permeating polar or large molecules into living cells. In the course of our study aiming at evaluating PTD, we unexpectedly found that the fluorescent-dye-labeled glutathione S-transferase (GST) from Schistosoma japonicum without known PTDs was delivered into COS7 cells. The intracellular transduction of GST was also observed in HeLa, NIH3T3, and PC12 cells, as well as in hippocampal primary neurons, indicating that a wide range of cell types is permissive for GST transduction. Furthermore, we showed that the immunosuppressive peptide VIVIT fused with GST successfully inhibits NFAT activation. These results suggest that GST is a novel PTD which may be useful in the intracellular delivery of biologically active molecules, such as small-molecule drugs, bioactive peptides, or proteins.     

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.