Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana

Met-overaccumulating mutants provide a powerful genetic tool for examining both the regulation of the Met biosynthetic pathway and in vivo developmental responses of gene expression to altered Met levels. We have previously reported the identification of two Arabidopsis thaliana Met over-accumulation (mto) mutants, mto1-1 and mto2-1, that carry mutations in the genes encoding cystathionine gamma-synthase (CGS) and threonine synthase (TS), respectively. A third mutant, mto3-1, has recently been reported to carry a mutation in the gene encoding S-adenosylmethionine synthetase 3 (SAMS3). Here, we report the isolation of a new ethionine-resistant A. thaliana mutant that over-accumulates soluble Met approximately 20-fold in young rosettes. The causal mutation was determined to be a single, recessive mutation that was mapped to chromosome 3. Sequence analysis identified a single nucleotide change in the gene encoding SAMS3 that was distinct from the mto3-1 mutation and altered the amino acid sequence of the enzyme active site. This mutation was therefore referred to as mto3-2. Although Met over-accumulation in the mto3-2 mutant was similar to that in the mto2-1 mutant, CGS mRNA levels did not respond to the mto3-2 mutation and were similar to that in equivalent wild-type plants.     

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.     

Planarian pharynx regeneration revealed by the expression of myosin heavy chain-A

The pharynx is a distinctive organ in the center of the body of planarians. Although the process of pharynx regeneration has been studied previously, the details and mechanism of the process remain controversial. We examined the process of regeneration of the pharynx in the planarian Dugesia japonica in detail by in situ hybridization and immunohistochemistry for myosin heavy chain-A (DjMHC-A), which is mainly expressed in the pharynx muscles and pharynx-anchoring muscles. We also monitored the behavior of the neoblasts in this process. In the regenerating posterior body fragment, the pharyngeal rudiment was formed by accumulation of cells that were probably undifferentiated cells derived from the neoblasts. The pharynx muscles appeared to differentiate in the rudiment in a manner that was coordinated with the differentiation of the pharynx-anchoring muscles in the region surrounding the rudiment. During this process, all cells containing mRNA for DjMHC-A also contained the DjMHC-A protein. These results argue against a previously proposed hypothesis that in the mesenchyme, 'pharynx-forming cells', which are committed to differentiate into the pharyngeal cells but have not yet differentiated, gather in the rudiment to form the pharynx (Agata and Watanabe, 1999). Rather, the present observations suggest that regeneration of the planarian pharynx proceeds by accumulation of cells that are probably undifferentiated cells derived from neoblasts in the rudiment, followed by their differentiation into the pharyngeal cells there.     

Variation in mitochondrial DNA of Vietnamese pigs: relationships with Asian domestic pigs and Ryukyu wild boars

Mitochondrial DNA (mtDNA) sequences (574 bp) of 30 Vietnamese pigs (large and small) were examined and compared with those of 61 haplotypes from wild boars and domestic pigs from various locations in Asia. The large Vietnamese pigs had genetic links to Ryukyu wild boars in southern Japan. The small Vietnamese pigs were closely related to other East Asian domestic pigs. These results indicate that Vietnamese pigs are genetically diverse and may be descendents of wild and domestic pigs from other regions of Asia.     

Analysis of gene expression profile in p130(Cas)-deficient fibroblasts

p130(Cas) (Cas) is a docking protein that becomes tyrosine phosphorylated in v-Src- or v-Crk-transformed cells and in integrin-stimulated cells. Cas -/- fibroblasts show defects in stress fiber formation, cell spreading, cell migration, and transformation by activated Src. To further characterize the role of Cas in signaling, we compared the expression profile in Cas -/- fibroblasts with that in Cas-re-expressing fibroblasts using the microarray methods. In Cas -/- fibroblasts, the expression of heme oxygenase 1 and caveolin-1 was reduced, but the expression of procollagen 1 alpha 1, procollagen 3 alpha 1, procollagen 11 alpha 1, elastin, periostin, TSC-36, and MARCKS was enhanced. The domains in Cas necessary for the change varied among these genes. Activated Src reduced the expression of most of these genes both in Cas -/- and in Cas +/+ fibroblasts. These results suggest the existence of signaling pathways that emanate from Cas to gene expression.     

Dual abnormal effects of mutant MITF encoded by Mi(wh) allele on mouse mast cells: decreased but recognizable transactivation and inhibition of transactivation

Structural localization of a peptide region, KRQPRNPKTDKLVNE, in the catalytic subunit of (Na(+) + K(+))-ATPase was investigated using a specific antibody directed against this peptide in cultured African green monkey kidney CV-1 cells. Immunofluorescence staining of frozen cell sections shows that an anti-KRQPRNPKTDKLVNE antibody (SSA95) interacts with its antigenic site and binds to the extracellular side of the cell membrane. Indirect immunofluorescence and flow cytometric analyses confirmed the presence of this epitope on intact cell surfaces. These results suggest that the KRQPRNPKTDKLVNE region of the (Na(+) + K(+))-ATPase is expressed on the cellular membrane surface.     

A new technique to co-localise membrane proteins with Homer/vesl

The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.