1-beta-O-Acyl glucuronides (betaGAs) are potentially reactive metabolites capable of binding to proteins, and they have been implicated in adverse drug reactions of the carboxylic acid drugs. To explore their electrophilic reactivity, we studied structure-activity relationships (SARs) to characterize the factors affecting the degradation rate constants (k values) of betaGAs and ultimately to predict k values of structurally diverse betaGAs. Twenty-seven betaGAs and four related compounds were synthesized, and their k values were determined under physiological conditions (pH 7.4 and 37 degrees C). 1-beta-O-Benzoyl glucuronide (BAGA) and glucopyranoside (BAG) showed almost the same k values, whereas their 1-alpha-O-benzoyl isomers degraded approximately 40-fold faster than BAGA and BAG. BAGA methyl ester showed almost the same rate constant as BAGA in the cleavage of their 1-beta-O-benzoyl linkages. A pH-log k profile obtained indicated kinetics catalyzed by both specific and general bases. The log k of betaGAs derived from m- and p-substituted benzoic acids correlated with Hammett's sigma constants. A similar correlation was observed with delta(COOH), (1)H NMR chemical shifts of the parent benzoic acids including ones with less sterically bulky o-substituents. Alternative descriptors of delta(CO) and delta((CO)OH), (13)C chemical shifts for ester carbonyl carbons of betaGAs and for carbonyl carbons of the parent benzoic acids, respectively, correlated well with the log k of all 16 betaGAs derived from benzoic acids including ones with bulkier o-substituents. Of the betaGA isomers derived from (2R)- and (2S)-alpha-methyl-4-biphenylylacetic acid, the (2R)-isomer degraded approximately 2-fold faster than the (2S)-isomer. The alpha-methyl group in the (2S)-isomer would encumber the intramolecular acyl migration. The log k of betaGAs derived from n-aralkyl carboxylic acids and of the (2R)-isomer correlated with their delta(COOH). However, the log k of betaGAs derived from alpha,alpha-dimethyl- and alpha,alpha-diethyl-4-biphenylylacetic acids deviated downward from the regression line, probably due to a steric effect. The diversity and complexity of k values were discussed with respect to the electrophilicity of the ester carbonyl carbons of betaGAs and the steric hindrance around them.

Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin␣, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin ␣ homologue from rice which is in addition to our previously isolated rice importin ␣1a and ␣2, and we have named it rice importin ␣1b. In vitro binding and nuclear import assays using recombinant importin ␣1b protein demonstrate that rice importin ␣1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin ␣ genes revealed that the genes were not only differentially expressed but that they also responded to darkadaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin ␣1b. These data suggest that each of the different rice importin ␣ proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin ␣ contribute to the regulation of plant nuclear protein transport.The most characteristic feature of an eukaryotic cell is the presence of a nuclear envelope, which separates the cell into two major compartments, the nucleus and the cytoplasm. Communication between these two compartments takes place through the nuclear pore complex (NPC) 1 (for review, see Refs.1-3). The NPC allows molecules smaller than 40 -60 kDa to diffuse across, while larger proteins and RNA-protein complexes must be actively transported through the NPC in a signal-mediated and energy-dependent manner. Nuclear proteins involved in nuclear activities, such as DNA replication, transcriptional RNA synthesis, and RNA splicing, must enter into the nucleus. Conversely, RNA, such as mRNA synthesized in the nucleus, must be transported into the cytoplasm where it is translated to protein. In plants, nucleocytoplasmic transport has been implicated in functional regulation of a number of plant photomorphogenesis related protein factors (4). For example, constitutive photomorphogenic 1 (COP1), a repressor of photomorphogenesis, has been shown to shuttle between the nucleus and cytoplasm in response to a change of light environment. COP1 exists predominantly in the cytoplasm in the light while it accumulates in the nucleus in the dark, suggesting that nuclear protein transport is an underlying mechanism for the regulation of COP1 activity (5). Multiple pathways of nucleocytoplasmic transport have been identified, each likely to be involved in carrying a distinct group of proteins (for review, see Refs. 3 and 6). Among them, the best characterized is the import of proteins containing a classical nuclear localization signal (NLS) that consists of either a short stretch of 3-5 basic amino acids or two basic domains separated by a spacer, referred to as monopartite and bipartite NLS, respectively (7). Yeast mating factor (Mat␣-2) contains a NLS consisting of basic and hydrophobic amino acid residues and has also been shown to...

Using a straightforward chemo-enzymatic procedure, 1-beta-O-acyl glucuronides of three non-steroidal anti-inflammatory drugs, diclofenac (DF) 5, mefenamic acid (MF) 6 and (S)-naproxen (NP) 7, were prepared. Caesium salts of these carboxylic acid drugs reacted with commercially available methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate 4 to give exclusively the corresponding 1-beta-O-acyl glucuronides 8-10 in moderate yields. The protecting acetyl (for -OH group) and methyl ester (for -CO2H group) groups of each sugar moiety were easily removed to provide the corresponding free 1-beta-O-acyl glucuronides 1-3 in high yields. Deprotection was achieved through effective enzyme-catalysed chemo-selective hydrolyses of the acetyl groups using lipase AS Amano (LAS), and of the methyl ester group using esterase from porcine liver (PLE).

Since 1-beta-O-acyl glucuronides (betaGAs) are thought to be chemically reactive metabolites capable of binding to tissue proteins, possibly leading to adverse drug reactions of the parent carboxylic acid drugs, we have initiated research efforts to derive structure-activity relationships (SARs) of betaGAs, with a focus on finding appropriate descriptors that predict their intrinsic electrophilic reactivity or degradation rate constants (k values). Our previous SAR studies on the k values of betaGAs derived from o-, m-, and p-substituted benzoic acids demonstrated that the diversity and complexity of the k values were controlled by the electronic and/or steric effects of the parent carboxylic acids. In the present study, we performed further SAR studies on the k values of 13 betaGAs derived from aralkyl carboxylic acids, focusing on the substituents and stereochemistry at the alpha-position of the parent carboxylic acids. In single regression analyses, the pKa and (1)H NMR chemical shifts (delta(COOH)) of the parent carboxylic acids correlated well with the log k values of seven betaGAs derived from five arylacetic and two (R)-2-arylpropionic acids, whereas the (13)C NMR chemical shifts [delta(C horizontal lineO) and delta((C horizontal lineO)OH)] correlated with the log k values of another seven betaGAs derived from the five arylacetic and two (S)-2-arylpropionic acids. Excellent correlations were also obtained between the log k values of four betaGAs with a common 4-phenylbenzyl moiety and the partial atomic charges (natural type) of the corresponding carboxylic hydrogen atoms (Hpac), the molar volume (MV), and the molar refractivity (MR). In multiple regression analyses, appropriate combinations of electronic (delta(COOH) or pKa) and steric [Taft's steric constant (Es) or delta((C horizontal lineO)OH)] descriptors could predict the log k values of betaGAs; electron-withdrawing 1-beta-O-acyl groups increased the k values, while increasing steric hindrance around the linkages decreased them. The standard partial regression coefficients indicated that the steric effects of the 1-beta-O-acyl groups of betaGAs affected the k values as strongly as the electronic effects. External validation of the derived SAR models is also discussed.

Conclusion:Otolith dysfunction occurred in acute attacks in a considerable number of patients with Meniere's disease. SVV can be utilized as a good tool for the evaluation of otolith dysfunction at acute attacks in patients with Meniere's disease.