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Plasma concentration profiles of arundic acid ((R)-(-)-2-propyloctanoic acid), an oil-like medicine, administered as soft-gel capsules in human clinical tests were predicted from the dissolution test data of the soft-gel capsules with different storage terms (short- and long-term stored drugs) by applying the in vitro-in vivo correlation (IVIVC). We established two linear-regression IVIVCs, which were characterized by either the in vitro dissolution behaviors against the pH 8.0 dissolution medium or the pH 6.8 dissolution medium containing 2% sodium dodecyl sulfate (SDS), in this study. Also, the prediction accuracies for the in vivo plasma profiles in humans for these two IVIVCs were compared. Regarding dissolution from the long-term stored capsule in pH 8.0 dissolution medium without surfactant, the prediction accuracies of the in vivo plasma profiles in humans were not satisfactory for the obtained IVIVC. The use of pH 6.8 dissolution medium containing 2% SDS, according to the Japanese guideline, improved the dissolution of the long-term stored capsule. Furthermore, the prediction accuracies for the in vivo plasma profiles in humans for these two IVIVCs were compared. The IVIVC established by the in vitro dissolution data obtained with the dissolution medium containing surfactant more effectively predicted the plasma drug concentration profiles following oral administrations of the soft-gel capsules under both storage conditions.

A highly sensitive and selective method has been developed and validated to determine limaprost, a prostaglandin (PG) E(1) analogue, in human plasma by on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry (2D-LC/MS/MS) due to the lack of efficient methods to determine very low levels of limaprost in plasma. Limaprost and its deuterium derivatives, used as internal standard, were extracted by protein precipitation and following three-step solid phase extractions. After extraction procedure, samples were analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system consists of Phenyl column at first dimension and ODS at second dimension with a trapping column placed between the separation columns. The linear dynamic range of this method was 0.1-10 pg/ml with 3 ml of plasma (r >0.9987). Acceptable precision and accuracy were obtained over the calibration curve ranges. The assay has been successfully used in analyses of human plasma samples to support clinical pharmacokinetics studies.

This study aimed to assess the steady-state pharmacokinetics of pranlukast, a leukotriene receptor antagonist, in children with allergic rhinitis and bronchial asthma, and to clarify factors affecting apparent clearance (CL/F). A total of 192 plasma samples were obtained from 98 children (rhinitis 64, asthma 13, complications 21), aged 3-14 years in 2 clinical trials. Plasma concentration of pranlukast was determined by liquid chromatography connected with a tandem mass spectrometer and analyzed by a population approach using NONMEM program. The plasma concentration-time course of pranlukast was described by using a one-compartment model with the first-order absorption and lag time. The robustness of the population pharmacokinetic model was evaluated by using 200 bootstrap samples. The results of population pharmacokinetic analysis showed that only age was a factor affecting the CL/F per body weight, with CL/F decreasing with increasing age. No significant variation was seen in the CL/F between rhinitis and asthma. The interindividual variability in the CL/F and the residual variability were 19.7% and 48.4%, respectively. All the parameters fell within 10% of the bootstrapped mean. In conclusion, the results show that age is the most influential factor for explaining interindividual variability in CL/F, and the difference in diseases does not affect CL/F.

Electron lifetime (tau) in dye-sensitized solar cells (DSC), using electrolytes containing I-/I3- redox couple, was investigated with quaternary ammonium cations having various alkyl chain lengths, and it was found that the lifetime increased in the order tau(TPA) approximately tau(TBA) < tau(THA) approximately tau(THpA), where tau(TPA), tau(TBA), tau(THA) and tau(THpA) were the lifetimes with tetra-propyl, butyl, hexyl and heptyl ammonium cations respectively. On the other hand, little influence of the cations on electron diffusion coefficients was observed, indicating that the DSCs using these cations have longer electron diffusion length (L). New electrolyte compositions for DSCs were sought with THA+ to increase the lifetime and L without compromising charge injection yield from the dye to TiO2. The electrolyte was applied for DSCs using TiO2 electrode prepared by 150 degrees C sintering. Under one-sun conditions (100 mW cm(-2)), newly prepared electrolytes showed at most 40% increase of energy conversion efficiency in comparison to conventional electrolytes and the highest value was 4.7%, which was the highest among the reported values of low temperature processed DSCs. The increase of the efficiency was achieved by higher short-circuit current induced by longer electron diffusion length and higher open-circuit voltage due to higher electron density.

The performance of dye-sensitized solar cells (DSCs) was compared before and after processing the TiO(2) electrodes by minute-order electrochemical reactions with metal nitrates, where the metals were Mg, Zn, Al, and La, in 2-propanol. An overcoating of metal hydroxide was formed without the need for a sintering process, and magnesium hydroxide was found to give the largest improvement in photovoltage, fill factor, and eventually overall conversion efficiency of the DSCs. To analyze the nature of the improvement, the diffusion coefficient (D) and electron lifetime (tau) were determined. While little influence of overcoating on D was seen, a correlation between the increase in tau and V(oc) was observed for the metals examined here. The remarkable improvement in the electron lifetime of the DSCs suggests that an overcoating with magnesium hydroxide species function as the blocking layers at the fluorine-doped tin oxide and TiO(2) interfaces, thus contributing to the suppression of electron leakage, i.e., recombination processes between unidirectional transporting electrons and poly-iodides such as tri-iodide in the processed TiO(2) photoelectrode systems. The increase in V(oc) can be explained by the increased electron density caused by the increase in electron lifetime.

Various countries have established regulations that stipulate the labeling of agricultural commodities, feed, and food products that contain or are made from genetically modified (GM) material or that contain adventitious GM material in amounts that exceed certain threshold levels. While regulations in some countries refer to GM material on a weight per weight (w/w) percentage, the currently applied detection methods do not directly measure the w/w percentage of the GM material. Depending on the particular method and the sample matrix it is applied to, the conversion of analytical results to a w/w percentage is challenging or not possible. The first rapid PCR system for GM maize detection on a single kernel basis has been developed. The equipment for the grinding of individual kernels and a silica membrane-based 96-well DNA extraction kit were both significantly revised and optimized for this particular purpose, respectively. We developed a multiplex real-time PCR method for the rapid quantification of GM DNA sequences in the obtained DNA solutions. In addition, a multiplex qualitative PCR detection method allows for the simultaneous detection of different GM maize traits in each kernel and thereby for identification of individual kernels that contain a combination of two or more GM traits. Especially for grain samples that potentially contain combined-trait GM maize kernels, the proposed methods can deliver informative results in a rapid, precise, and reliable manner.

Performance of dye-sensitized solar cells (DSCs) was investigated depending on the compositions of the electrolyte, i.e., the electrolyte with a different cation such as Li(+), tetra-n-butylammonium (TBA(+)), or 1,2-dimethyl-3-propylimidazolium (DMPIm(+)) in various concentrations, with and without 4-tert-butylpyridine (tBP), and with various concentrations of the I(-)/I(3)(-) redox couple. Current-voltage characteristics, electron lifetime, and electron diffusion coefficient were measured to clarify the effects of the constituents in the electrolyte on the charge recombination kinetics in the DSCs. Shorter lifetimes were found for the DSCs employing adsorptive cations of Li(+) and DMPIm(+) than for a less-adsorptive cation of TBA(+). On the other hand, the lifetimes were not influenced by the concentrations of the cations in the solutions. Under light irradiation, open-circuit voltages of DSCs decreased in the order of TBA(+)> DMPIm(+) > Li(+), and also decreased with the increase of [Li(+)]. The decreases of open-circuit voltage (V(oc)) were attributed to the positive shift of the TiO(2) conduction band potential (CBP) by the surface adsorption of DMPIm(+) and Li(+). These results suggest that the difference of the free energies between that of the electrons in the TiO(2) and of I(3)(-) has little influence on the electron lifetimes in the DSCs. The shorter lifetime with the adsorptive cations was interpreted with the thickness of the electrical double layer formed by the cations, and the concentration of I(3)(-) in the layer, i.e., TBA(+) formed thicker double layer resulting in lower concentration of I(3)(-) on the surface of the TiO(2). The addition of 4-tert-butylpyridine (tBP) in the presence of Li(+) or TBA(+) showed no significant influence on the lifetime. The increase of V(oc) by the addition of tBP into the electrolyte containing Li(+) and the I(-)/I(3)(-) redox couple was mainly attributed to the shift of the CBP back to the negative potential by reducing the amount of adsorbed Li cations.

Dye-sensitized solar cells (DSC) were prepared from nanoporous TiO(2) electrodes with two different cobalt complex redox couples, propylene-1,2-bis(o-iminobenzylideneaminato)cobalt(II) {Co(II)(abpn)} and tris(4,4'-di-tert-buthyl-2,2'-bipyridine)cobalt(II) diperchlorate {Co(II)(dtb-bpy)(3)(ClO(4))(2)}. The performances of the DSCs were examined with varying the concentrations of the redox couples and Li cations in methoxyacetonitrile. Under 1 sun conditions, short-circuit currents (J(sc)) increased with the increase of the redox couple concentration, and the maximum J(sc) was found at the Li(+) concentration of 100 mM. To rationalize the observed trends of J(sc), electron diffusion coefficients and lifetimes in the DSCs were measured. Electron diffusion coefficients in the DSCs using cobalt complexes were comparable to the previously reported values of nanoporous TiO(2). Electron lifetime was independent of the concentration of the redox couples when the concentration ratio of Co(II)(L) and Co(III)(L) was fixed. With the increase of Li(+) concentration, the electron lifetime increased. These results were interpreted as due to their slow charge-transfer kinetics and the cationic nature of Co complex redox couples, in contrast to the anionic redox couple of I(-)/I(3)(-). The increase of the lifetimes with Li(+) was interpreted with the decrease of the local concentration of Co(III) near the surface of TiO(2). The addition of 4-tert-butylpyridine (tBP) with the presence of Li(+) increased J(sc) significantly. The observed increase of the electron lifetime by tBP could not explain the large increase of J(sc), implying that tBP facilitates the charge transfer from Co(II)(L) to dye cation, with the association of the change of the reorganization energy between Co(II) and Co(III).

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.

A population pharmacokinetic substudy design of a new chemical entity was evaluated based on the bias in parameter estimates and the power of detecting a specific subpopulation showing different clearance using a clinical trial simulation approach. The effect of analysis algorithms on type I error was also assessed. The design factors included the number of patients (n=100-300) and the number of sampling points per patient (n=2-6). Simulation data were generated from a model developed based on a Phase I study. The power was evaluated for a percentile of test statistics obtained by the simulation study. The clearance (CL) related parameters were estimated with sufficient accuracy in all study designs and all analysis algorithms: the first order (FO), first order conditional estimation (FOCE) and first order conditional estimation with interaction (FOCE-INTER) methods. With the FO and FOCE methods, the type I error rate increased as the frequency of sampling from each patient became higher, but such increase was hardly observed with the FOCE-INTER method. The power tended to depend on the size of the subpopulation. A large difference was found in the power of detecting a specific subpopulation showing a clearance decrease of 30% or 50%. Therefore, the most dominant factors controlling power would be the size of the subpopulation and the decreasing ratio of CL in the subpopulation. These findings obtained by the clinical trial simulation approach are useful for optimization of study design and determination of the limits of evaluation.

The hypocalcemic effect of salmon calcitonin (sCT) after intravenous administration was explained on the basis of an integrated pharmacokinetic-pharmacodynamic (PK-PD) model with the endogenous Ca regulatory system in the rat. The pharmacokinetics of sCT described by a conventional two-compartment model showed the extremely rapid elimination of sCT from plasma (MRT; 6.86 min). The hypocalcemic effect of sCT reached a peak from 0.5 to 1.5 hrs after administration, and the peak time tended to prolong with increasing doses. This delay in pharmacological effect of sCT against plasma concentration may be a result of a summation of multiple actions of the endogenous Ca regulatory system including feedback control. The plasma Ca regulation system in the rat was investigated by i.v. bolus administration of calcium gluconate and/or endogenous (rat) calcitonin (rCT). Since non-linearity in the relationship between Ca and rCT concentrations in plasma was observed, we assumed that rCT was secreted in accordance with the plasma Ca level via an exponential function. The pharmacokinetics of rCT was represented as a linear one-compartment model. To link the rCT level with plasma Ca level, an additional effect compartment was required to explain the delay in onset and decline of the pharmacological effect. This Ca regulation model explained the observed Ca and rCT profiles in plasma after administration of Ca and/or rCT. The plasma Ca levels after administration of sCT could be well described by the present integrated model. This suggested the potential for prediction of plasma sCT concentration only from the hypocalcemic effect after extravascular administration of sCT, using this PK-PD model.

Assessment of the extent of bioavailability (EBA) of salmon calcitonin (sCT) from hypocalcemic effects after intranasal administration was presented in rats. An integrated pharmacokinetic-pharmacodynamic (PK-PD) model with the endogenous Ca regulation system was applied. The influence of camostat mesilate, a protease inhibitor, on absorption of sCT was also estimated. Camostat, coadministered intravascularly, delayed the elimination of sCT. Although the hypocalcemic effect of sCT after i.v. administration was accelerated when camostat was coadministered intravenously, the enhanced effect could not be expressed only by pharmacokinetic change of sCT, and then the pharmacological data in the presence of camostat were analyzed to obtain optimal PD parameters. For the absorption of sCT after i.n. administration, a saturable absorptive process and a zero-order kinetic clearance from the nasal cavity were introduced to the model. The regression curves fitted the observed data, and camostat caused both an increase in maximum absorption rate and a decrease in the clearance parameter compared with the control. According to this modified PK-PD relationship, plasma sCT concentrations following i.n. administration of sCT with camostat were predicted well using its pharmacological effects. The EBA of sCT calculated from the simulated concentrations increased more than 4-folds compared with the control study. These results indicate the potential for prediction of plasma sCT concentration from the hypocalcemic effect.

Background: HscC, the third member of the Hsp70 family in Escherichia coli, shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity. Genetic and biochemical evidence indicates that the DnaK-DnaJ chaperone system interacts with sigma32 and is involved in the negative regulation of the heat shock response. Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown., Results: We observed that the over-expression of HscC caused severe growth inhibition. To explore this effect, we performed primer extension analysis and a beta-galactosidase assay and found that HscC reduced the sigma70-dependent promoter activity. An in vitro transcription assay revealed that HscC inhibited sigma70-dependent transcription. In addition, the co-purification analysis showed that sigma70 co-eluted with HscC., Conclusion: These results indicate that HscC forms a complex with sigma70 and may function as its negative modulator.

From mouse genomic libraries and human brain cDNA, we cloned three novel G-protein-coupled receptors (GPCRs), which have about 55-70% homologies with Xenopus PSP24 (xPSP24). Together with another human cDNA (GPR45) cloned by Marchese et al. (Genomics 56, 12-21, 1999). they comprise a family of mammalian PSP24s. Therefore, we termed these clones mouse PSP24alpha, beta, and human PSP24alpha, beta. The homologies between alpha and beta isoforms were 54% for human and 51% for mouse clones. None of these clones shares sequence similarities with any known mammalian GPCRs, thus forming a unique gene family. Northern blot demonstrated that both of the mouse transcripts were predominantly expressed in the brain. In situ hybridization of brain sections showed that the expression was observed in neuronal cells, such as olfactory mitral cells, cortical neurons, hippocampal pyramidal cells, and Purkinje cells in the cerebellum. We suggest that mammalian PSP24 is a distinct GPCR family and plays a role in the brain function., (Copyright 2000 Academic Press.)

The contiguous 874.423 base pair sequence corresponding to the 50.0-68.8 min region on the genetic map of the Escherichia coli K-12 (W3110) was constructed by the determination of DNA sequences in the 50.0-57.9 min region (360 kb) and two large (100 kb in all) and five short gaps in the 57.9-68.8 min region whose sequences had been registered in the DNA databases. We analyzed its sequence features and found that this region contained at least 894 potential open reading frames (ORFs), of which 346 (38.7%) were previously reported, 158 (17.7%) were homologous to other known genes, 232 (26.0%) were identical or similar to hypothetical genes registered in databases, and the remaining 158 (17.7%) showed no significant similarity to any other genes. A homology search of the ORFs also identified several new gene clusters. Those include two clusters of fimbrial genes, a gene cluster of three genes encoding homologues of the human long chain fatty acid degradation enzyme complex in the mitochondrial membrane, a cluster of at least nine genes involved in the utilization of ethanolamine, a cluster of the secondary set of 11 hyc genes participating in the formate hydrogenlyase reaction and a cluster of five genes coding for the homologues of degradation enzymes for aromatic hydrocarbons in Pseudomonas putida. We also noted a variety of novel genes, including two ORFs, which were homologous to the putative genes encoding xanthine dehydrogenase in the fly and a protein responsible for axonal guidance and outgrowth of the rat, mouse and nematode. An isoleucine tRNA gene, designated ileY, was also newly identified at 60.0 min.

The 465,813 base pair sequence corresponding to the 40.1-50.0 min region on the genetic map of Escherichia coli K-12 (W3110) was determined. Analysis of the sequence revealed that this region contained at least 466 potential open reading frames, of which 187 (40%) were previously reported, 105 (23%) were homologous to other known genes, 103 (22%) were identical or similar to hypothetical genes registered in databases, and the remaining 71 (15%) did not show a significant similarity to any other gene. At the 45.2-46.0 min region, we found a very large cluster of about 30 genes, whose functions are involved in the biosynthesis of polysaccharides as the components of outer membranes. In addition, we identified a new asn-tRNA gene, designated asnW, between the asnT and asnU genes and a new lysogenic phage attachment site as the cis-element.

The PHO84 gene in Saccharomyces cerevisiae encodes a Pi transporter, mutation of which confers constitutive synthesis of repressible acid phosphatase (rAPase), in medium containing repressible amounts of Pi, and an arsenate-resistant phenotype. We selected an arsenate-resistant mutant showing the constitutive synthesis of rAPase on nutrient plates containing 4.5 mM arsenate. This mutant has double mutations designated as pho86 and pho87. The mutant transcribes PHO84 even in the repressible condition but has a severe defect in Pi uptake. The constitutive rAPase+ phenotype of the pho86 pho87 mutant was partially suppressed by an increased dosage of the PHO84 gene. The PHO87 gene was found to be identical with YCR524, according to the published nucleotide sequence of chromosome III, which encodes a protein of 923 amino-acid residues with a highly charged N-terminal half followed by a C-terminal half consisting of 12 membrane-spanning segments as in Pho84p. These and the other findings suggest that the Pho86p and Pho87p proteins collaborate with Pho84p in Pi uptake.

To study the role of the IP3 receptor (IP3R) upon egg activation, cDNA clones encoding IP3R expressed in the Xenopus oocytes were isolated. By analyses of the primary structure and functional expression of the cDNA, Xenopus IP3R (XIP3R) was shown to have an IP3-binding domain and a putative Ca2+ channel region. Immunocytochemical studies revealed polarized distribution of XIP3R in the cytoplasm of the animal hemisphere in a well-organized endoplasmic reticulum-like structure and intensive localization in the perinuclear region of stage VI immature oocytes. In ovulated unfertilized eggs, XIP3R was densely enriched in the cortical region of both hemispheres in addition to its polarized localization. After fertilization, XIP3R showed a drastic change in its distribution in the cortical region. These results imply the predominant role of the XIP3R in both the formation and propagation of Ca2+ waves at fertilization.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.

The concentration of cytoplasmic free calcium (Ca2+) increases in various stimulated cells in a wave (Ca2+ wave) and in periodic transients (Ca2+ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP3 receptor inhibited both IICR and CICR upon injection of IP3 and Ca2+ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. The results indicate that Ca2+ release in fertilized hamster eggs is mediated solely by the IP3 receptor, and Ca(2+)-sensitized IICR, but not CICR, generates Ca2+ waves and Ca2+ oscillations.

Inositol 1,4,5-trisphosphate (InsP3) receptor immunoreactivity in SH-SY5Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, approximately 275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half-maximal and maximal at approximately 2 and approximately 6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12-myristate 13-acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol-induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH-SY5Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releases Ca2+ from its intracellular stores. The InsP3 receptor has been purified and its cDNA has been cloned. We have found that the InsP3 receptor is identical to P400 protein, first identified as a protein enriched in cerebellar Purkinje cells. We have generated an L-fibroblast cell transfectant that produces cDNA-derived InsP3 receptors. The protein displays high affinity and specificity for InsP3. InsP3 induces greater Ca2+ release from membrane vesicles from transfected cells than from those from control L-fibroblasts. After incorporation of the purified InsP3 receptor into lipid bilayers InsP3-induced Ca2+ currents were demonstrated. These results suggest that the InsP3 receptor is involved in physiological Ca2+ release. Immunogold labelling using monoclonal antibodies against the receptor showed that it is highly concentrated on the smooth-surfaced endoplasmic reticulum and slightly on the outer nuclear membrane and rough endoplasmic reticulum; no labelling of Golgi apparatus, mitochondria and plasmalemma was seen. Cross-linking experiments showed that the receptor forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region contains the critical sequences for InsP3 binding. We have investigated the heterogeneity of the InsP3 receptor using the polymerase chain reaction and have found novel subtypes of the mouse InsP3 receptor that are expressed in a tissue-specific and developmentally specific manner.

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.

A mutant plasmid, pX, derived from the 1453 base pair small plasmid, YARp1 (or TRP1 RI circle), consists of 849 base pairs of DNA bearing the TRP1 gene and the ARS1 sequence of Saccharomyces cerevisiae and, unlike YARp1 and other commonly used yeast plasmids, highly multimerizes in a S. cerevisiae host. The multimerization of pX was dependent on RAD52, which is known to be necessary for homologous recombination in S. cerevisiae. Based upon this observation, a regulated system of multimerization of pX with GAL1 promoter-driven RAD52 has been developed. We conclude that the regulated multimerization of pX could provide a useful model system to study genetic recombination in the eukaryotic cell, in particular to investigate recombination intermediates and the effects of various trans-acting mutations on the multimerization and recombination of plasmids.