Improvement of LXR-mediated lipid metabolism in nephrotic model kidney accompanied by suppression of inflammation and fibrosis.

Kidney disease affects millions of people worldwide. Chronic kidney diseases, such as diabetic nephropathy, are often accompanied by nephrotic syndrome, which causes a large amount of protein and lipid to leak out into the urine. Leaked lipids are well known to accumulate in the proximal tubules as lipid droplets. However, the role of lipid metabolism in the kidney has not been thoroughly studied, and the relationship between accumulated lipid and pathological progression is often unknown. In this study, we showed that reducing accumulated lipids by exerting an agonistic effect on Liver X receptor, one of the nuclear receptors known to play an important role in lipid metabolism, suppressed the development of pathological conditions, such as inflammation and fibrosis, in a nephrosis model. Until now, many renal disease treatments have focused on suppressing the inflammatory response. However, it is now clear that even if the direct anti-inflammatory response is weak, the spread of inflammation and fibrosis can be suppressed by reducing accumulated lipids. Our results suggest that reducing abnormal lipid accumulation in the kidney could lead to disease treatment.

B-ring-homo-tonghaosu, isolated from Chrysanthemum morifolium capitulum, acts as a peroxisome proliferator-activated receptor-γ agonist.

The capitula of Chrysanthemum morifolium and C. indicum are used to prepare Chrysanthemi Flos in traditional Japanese Kampo medicine. In our previous study, we reported on the agonistic effect of methanol extract of C. indicum capitulum on peroxisome proliferator-activated receptor (PPAR)-γ. We further isolated (E)-tonghaosu from C. indicum capitulum as one of the active ingredients. In the present study, we aimed to evaluate the PPAR-γ agonistic activity of a methanol extract of C. morifolium capitulum (MCM) in which (E)-tonghaosu could not be detected. MCM exhibited PPAR-γ agonistic activity in a concentration-dependent manner, and at a dose of 100 µg/ml, it showed similar activity to pioglitazone (30 µM), a standard PPAR-γ agonist. Through activity-guided fractionation, we isolated two geometric isomers, (E)- (1) and (Z)-B-ring-homo-tonghaosu (2), as the active ingredients of MCM. Both compounds exerted concentration-dependent PPAR-γ agonistic effects, and 1 had higher activity than 2. At 1.4 µM, 1 had similar activity to pioglitazone (30 µM), which was achieved by 2 at a concentration of 140 µM. Thus, 1 has the potential to become a lead compound for the drug discovery of PPAR-γ agonists. We compared the activities and the contents of (E)-, (Z)-tonghaosu, 1, and 2 among 13 commercial samples of Chrysanthemi Flos, including those derived from both C. morifolium and C. indicum. Their PPAR-γ agonistic activities were not related to the contents of these compounds. 1 and 2 were detected in the samples derived from both species but (E)- and (Z)-tonghaosu were not detected in the samples derived from C. morifolium; hence (E)- and (Z)-tonghaosu can serve as marker compounds to identify the capitula of C. indicum in Chrysanthemi Flos samples.

Each liver X receptor (LXR) type has a different purpose in different situations.

LXRs, which are nuclear receptors, have 2 isoforms-LXRα and LXRß. Generally, LXRα is expressed in the liver, kidney, and a limited number of other organs, whereas LXRß is thought to be expressed ubiquitously. Nevertheless, no clear consensus has been reached on the role of each in kidney lipid metabolism. Many researchers have reported that lipids accumulate in renal tubular epithelial cells during nephrosis. The nephrosis model we used showed the presence of urinary protein 4 days after the induction of illness. Additionally, the model maintained high levels of urinary protein from day 7-14. Lipid accumulation was clearly verified at day 4 and extreme accumulation was observed at day 7. We observed increased expression of LXRα from an early stage of nephrosis. To explore the role of increased LXRα in diseased kidney in vitro, NRK52E, normal kidney tubular epithelial cells, were forced to overexpress LXRα. These cells showed significantly lower lipid accumulation than mock cells did. In contrast, LXRß knockdown lead to increased lipid accumulation in mock cells, and constancy in overexpressing cells. In normal kidneys, LXRß is expressed stably to control mainly the intracellular lipids. However, with increasing intracellular lipid accumulation, expression of LXRα and its downstream gene, ABCA1, was upregulated, followed by lipid excretion in an LXRα-dependent manner. This phenomenon strongly suggests the importance of LXRα in lipid metabolism in the diseased kidney.

Reduction of Membrane Protein CRIM1 Decreases E-Cadherin and Increases Claudin-1 and MMPs, Enhancing the Migration and Invasion of Renal Carcinoma Cells.

CRIM1 is a membrane protein that has been reported to be related to cell proliferation. CRIM1 is expressed in renal carcinoma cells, but its involvement in proliferation and malignant transformation remains unclear. We analyzed whether alterations in the characteristics of cancer cells are observed following knockdown of CRIM1. Decreased expression of CRIM1 did not affect proliferation or anchorage-independent growth. The results of wound healing and invasion assays showed that reduced expression of CRIM1 increased cells' migratory and invasive abilities. Expression analysis of factors involved in migration and invasion in CRIM1-knockdown cells revealed that expression of the cell adhesion factor E-cadherin declined and expression of claudin-1, which is upregulated in metastatic cancer cells, increased. In addition, increased expression of matrix metalloproteinase (MMP) 2 and MMP9, protease essential for cancer cell invasiveness, was observed. Furthermore, an increase in phosphorylated focal adhesion kinase (FAK), which increases cell migration, was observed. Increased expression of the E-cadherin transcription repressors Snail, Slug, and ZEB-1 were observed, and mRNA levels of E-cadherin were decreased. Therefore, expression of E-cadherin is thought to be decreased by both suppression of E-cadherin mRNA expression and promotion of degradation of the E-cadherin protein. In addition, expression of CRIM1 was decreased in renal cancer cells undergoing epithelial-mesenchymal transition (EMT) stimulated by tumor necrosis factor alpha (TNF-α). Thus, CRIM1 regulates the expression of several EMT-related factors and appears to play a role in suppressing migration and invasion through control of EMT.

Angiostatin prevents IL-1ß-induced down-regulation of eNOS expression by inhibiting the NF-κB cascade.

This study aimed to elucidate the protective potential of angiostatin in inflamed endothelial cells in culture. We assessed the effect of angiostatin on the expression of ICAM-1 and eNOS. Angiostatin prevented IL-1ß-induced down-regulation of eNOS expression, but produced no significant changes on IL-1ß-induced up-regulation of ICAM-1. We then explored the effect of angiostatin on IL-1ß-mediated inflammatory signaling and found that angiostatin inhibited IL-1ß-mediated nuclear translocation of NF-κB. Thus, our results suggest that angiostatin prevents IL-1ß-induced down-regulation of eNOS expression via inhibition of the NF-κB cascade; this may be the anti-inflammatory mechanism of angiostatin.

Implication of Akt-dependent Prp19 alpha/14-3-3beta/Cdc5L complex formation in neuronal differentiation.

PRP19alpha and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19 alpha/14-3-3beta/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Analysis of PRP19 alpha mutants revealed that the phosphorylation of PRP19 alpha at Thr 193 by Akt was critical for its binding with 14-3-3beta to translocate into the nuclei and for PRP19 alpha/14-3-3beta/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19 alpha or sense 14-3-3beta RNAs promoted NGF-induced neuronal differentiation, whereas down-regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19 alpha T193A lost its binding ability with 14-3-3beta and acted as a dominant-negative mutant in neuronal differentiation. These results imply that Akt-dependent phosphorylation of PRP19 alpha at Thr193 triggers PRP19 alpha/14-3-3beta/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre-mRNAs.

Angiostatin inhibition of vascular endothelial growth factor-stimulated nitric oxide production in endothelial cells.

Angiostatin (AS), a proteolytic fragment of plasminogen, is a potent antiangiogenic factor. It was reported that AS attenuates the vasodilatory response to vascular endothelial growth factor (VEGF) in isolated interventricular arterioles. Here, we investigated the effect of AS on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). AS inhibited VEGF-stimulated NO production in a dose-dependent manner, whereas AS alone did not affect basal NO production. Disruption of kringle structures by reduction of disulfide bonds resulted in the loss of the inhibitory effect of AS on VEGF-stimulated NO production. To elucidate how AS might impair VEGF activation of endothelial NO synthase (eNOS), we further examined whether AS would affect Ca(2+)-dependent and -independent pathways of eNOS activation. AS had no effect on the transient increase in cytosolic Ca(2+) levels elicited by VEGF. In contrast, AS prevented VEGF-potentiated eNOS phosphorylation at Ser1177. These results clearly indicate that AS inhibits VEGF-stimulated NO production in HUVECs without affecting basal NO production. The kringle structures of AS are required for this effect, and impairment of Ser1177 phosphorylation of eNOS might be involved in the inhibition of VEGF-stimulated NO production by AS.

A novel binding factor of 14-3-3beta functions as a transcriptional repressor and promotes anchorage-independent growth, tumorigenicity, and metastasis.

The 14-3-3 proteins form a highly conserved family of dimeric proteins that interact with various signal transduction proteins and regulate cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the beta isoform of 14-3-3 proteins promotes tumorigenicity and angiogenesis of rat hepatoma K2 cells. In this study, to analyze the mechanism of 14-3-3beta-induced malignant transformation, yeast two-hybrid screening was performed, and a novel 14-3-3beta-binding factor, FBI1 (fourteen-three-three beta interactant 1), was identified. In vitro binding and co-immunoprecipitation analyses verified specific interaction of 14-3-3beta with FBI1. The strong expression of FBI1 was observed in several tumor cell lines but not in non-tumor cell lines. Forced expression of antisense FBI1 in K2 cells inhibited anchorage-independent growth but had no significant effect on cell proliferation in monolayer culture. Down-regulation of FBI1 also inhibited tumorigenicity and metastasis accompanying a decrease in MMP-9 (matrix metalloproteinase-9) expression. In addition, the duration of ERK1/2 activation was curtailed in antisense FBI1-expressing K2 cells. A luciferase reporter assay revealed that the FBI1-14-3-3beta complex could act as a transcriptional silencer, and MKP-1 (MAPK phosphatase-1) was one of the target genes of the FBI1-14-3-3beta complex. Moreover, chromatin immunoprecipitation analysis demonstrated that FBI1 and 14-3-3beta were presented on the MKP-1 promoter. These results indicate that FBI1 promotes sustained ERK1/2 activation through repression of MKP-1 transcription, resulting in promotion of tumorigenicity and metastasis.

Involvement of the mouse Prp19 gene in neuronal/astroglial cell fate decisions.

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.

Polymorphism of dopamine receptor D4 exon I corresponding region in chicken.

In stockbreeding, there are indications that behavioral traits of livestock have an effect on breeding and production. If the variation in individual behavior is related to that in neurotransmitter-related genes such as in humans, it would be possible to breed pedigrees composed of individuals having behavioral traits that are useful to production and breeding using selection based on genotypes. In this study, we investigated the exon I region of dopamine receptor D4 (DRD4), in which variation is related to psychiatric disorder in humans, in major poultry species namely Japanese quail (Coturnix japonica), chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and helmeted guinea fowl (Numida meleagris). Furthermore, we investigated Japanese cormorant (Phalacrocorax capillatus) and Japanese jungle crow (Corvus macrorhynchos) as an out-group. In these species of birds, the repeat of proline was identified in the region corresponding to the human polymorphic region. The repeat number was 9 in Japanese quail, ring-necked pheasant and Japanese cormorant; 12 in helmeted guinea fowl; and 3 in Japanese jungle crow. However, no polymorphism was found in these species. In contrast, polymorphism was observed in chicken and two alleles with 8 and 9 repeats were identified. Although 9 repeats (allele 9) were predominant in most chicken breeds, Black Minorca had only 8 repeats (allele 8). Intra-breed polymorphism was found in 6 out of 12 breeds, and two alleles (alleles 8 and 9) were detected in these breeds. This polymorphism, which is the first to be reported on a neurotransmitter-related gene in birds, would contribute significant information for elucidation of differences in behavioral traits in chicken breeds.

Roles of poly(3-hydroxybutyrate) depolymerase and 3HB-oligomer hydrolase in bacterial PHB metabolism.

Many poly-3-hydroxybutyrate (PHB)-degrading enzymes have been studied. But biological roles of 3HB-oligomer hydrolases (3HBOHs) and how PHB depolymerases (PHBDPs) and 3HBOHs cooperate in PHB metabolism are not fully elucidated. In this study, several PHBDPs and 3HBOHs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. From the results, PHBDP and 3HBOH seemed to play a role in PHB metabolism in three types of bacteria, as follows: (A) In Ralstonia pickettii T1, an extracellular PHBDP degrades extracellular PHB to various-sized 3HB-oligomers, which an extracellular 3HBOH hydrolyzes to 3HB-monomers. (B) In Acidovorax sp. SA1, an extracellular PHBDP hydrolyzes extracellular PHB to small 3HB-oligomers (dimer and trimer), which an intracellular 3HBOH efficiently degrades to 3HB in the cell. (C) In Ralstonia eutropha H16, an intracellular 3HBOH helps in the degradation of intracellular PHB inclusions by PHBDP.

Forced expression of antisense 14-3-3beta RNA suppresses tumor cell growth in vitro and in vivo.

The 14-3-3 family proteins are key regulators of various signal transduction pathways including malignant transformation. Previously, we found that the expression of the 14-3-3beta gene is deregulated as well as c-myc gene in aflatoxin B1 (AFB1)-induced rat hepatoma K1 and K2 cells. To elucidate the implication of 14-3-3beta in tumor cell growth, in this paper we analyzed the effect of forced expression of antisense 14-3-3beta RNA on the growth and tumorigenicity of K2 cells. K2 cells transfected with antisense 14-3-3beta cDNA expression vector diminished their growth ability in monolayer culture and in semi-solid medium. Expression level of vascular endothelial growth factor mRNA was also reduced in these transfectants. Tumors that formed by the transfectants in nude mice were much smaller and histologically more benign tumors, because of their decreased level of mitosis compared with those of the parental cells. Frequency of apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was increased in the transfectant-derived tumors accompanying the inhibition of angiogenesis. In addition, over-expression of 14-3-3beta mRNA was observed in various murine tumor cell lines. These results suggest that 14-3-3beta gene plays a pivotal role in abnormal growth of tumor cells in vitro and in vivo.

Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase.

An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2(Reu)) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2(Reu). The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1(Reu)). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.

The role of transcriptional corepressor Nif3l1 in early stage of neural differentiation via cooperation with Trip15/CSN2.

Mouse Nif3l1 gene is highly conserved from bacteria to human. Even though this gene is expressed throughout embryonic development, its biological function is still obscure. Here, we show that Nif3l1 participates in retinoic acid-primed neural differentiation of P19 embryonic carcinoma cells through cooperation with Trip15/CSN2, a transcriptional corepressor/component of COP9 signalosome. We isolated Nif3l1 cDNA from P19 cell cDNA library by a yeast two-hybrid screening using Trip15/CSN2 as a bait. This interaction was confirmed by a pull-down assay and an epitope-tagged coimmunoprecipitation. Although Nif3l1 was mainly detected in the cytoplasm, the translocation of Nif3l1 into the nuclei was observed in retinoic acid-primed neural differentiation of P19 cells and enhanced by the enforced expression of Trip15/CSN2. Furthermore, enforced expression of sense Nif3l1 RNA, but not antisense RNA, enhanced the neural differentiation of P19 cells accompanying the intense down-regulation of Oct-3/4 mRNA expression and the rapid induction of Mash-1 mRNA expression. Luciferase reporter assay showed that Nif3l1 could act as a transcriptional repressor and synergized the transcriptional repression by Trip15/CSN2. These results indicate that Nif3l1 implicates in neural differentiation through the cooperation with Trip15/CSN2.

Implication of Trip15/CSN2 in early stage of neuronal differentiation of P19 embryonal carcinoma cells.

Trip15/CSN2 is a transcriptional corepressor/a component of COP9 signalosome (CSN) and participates in various signaling pathways. However, participation of Trip15/CSN2 in neural differentiation is still obscure. Here, we show that Trip15/CSN2 plays a critical role in neuronal differentiation. The expression of Trip15/CSN2 mRNA was induced at an early stage of neuronal differentiation in the retinoic acid (RA)-treated P19 cells, but not in the triiodothyronine (T3)-primed cardiac muscular cell differentiation. The expression of Trip15/CSN2 mRNA in the rat brain was detected at E14 and the protein was localized in the nuclei of neonatal rat CNS neurons. Enforced expression of sense rat Trip15/CSN2 mRNA caused the downregulation of Oct-3/4 mRNA expression and was sufficient to convert P19 cells into neurons, but not glial cells, only after the aggregation without RA. In the presence of RA, exogenous expression of the sense mRNA caused the intense and rapid induction of neurogenic Brn-2 and Mash-1 mRNA expressions accompanying the strong downregulation of Oct-3/4 mRNA expression, and stimulated both neuronal and glial cell differentiations of P19 cells. In contrast, enforced expression of the antisense mRNA suppressed the commitment of RA-treated aggregation form of P19 cells to neuronal lineage. These data strongly suggest that Trip15/CSN2 could implicate in the commitment of multipotent embryonal carcinoma (EC) cells to neuronal fate through the downregulation of Oct-3/4 which suppresses neurogenic genes. Moreover, in addition to Trip15/CSN2, RA-regulated other factor(s) may be required for glial cell differentiation.

Sodium 5, 6-benzylidene-L-ascorbate induces in vitro neuronal cell differentiation accompanying apoptosis and necrosis.

Antiproliferative activity through induction of differentiation by chemotherapeutic agents is required for certain types of cancers. Here, we report that a potent antitumor agent, sodium 5, 6-benzylidene-L-ascorbate (SBA), could induce morphological change of human neuroblastoma IMR-32 cells into a ganglion-like cell aggregate (pseudoganglion) having many neurites and the property of cholinergic neurons. Simultaneously with neuronal differentiation, substantial apoptosis and necrosis/type 2 physiological cell death, which is independent of apoptosis and resistant to a broad-spectrum caspase inhibitor, Z-Asp-CH2-DCB, were also observed. These data indicated that SBA could suppress tumor cell growth through the induction of three different physiological pathways such as differentiation, apoptosis and necrosis by which tissues and organs regulate their own development and maintenance.

Cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Acidovorax sp. strain SA1 and purification of the enzyme.

The gene of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase (i3HBOH) was cloned and sequenced from a poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Acidovorax sp. strain SA1. The i3HBOH gene has 876 nucleotides corresponding to the deduced sequence of 292 amino acids. In this amino acid sequence, the general lipase box sequence (G-X(1)-S-X(2)-G) was found, whose serine residue was determined to the active sites serine by site-directed mutagenesis. An i3HBOH was purified to electrophoretical homogeneity from SA1. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE. The N-terminal amino acid sequence of the purified enzyme corresponded to the deduced N-terminal amino acid sequence in the cloned i3HBOH gene. This is the first cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase gene to date.

Intrathecal high-dose morphine induces spinally-mediated behavioral responses through NMDA receptors.

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl-D-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D-APV) (6.25-50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125-25.0 pmol). The characteristic behavior was also reduced by co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (74.1-250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive antagonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of D-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.

Development of novel whole-mount in situ hybridization (WISH).

We have designed a novel fluorescent PNA probe which can be rapidly introduced into the living cell without any complicated pretreatment. We could post-synthetically incorporate amino acid derivatives having membrane permeability function into the PNA probe by utilizing key compound 1 which we have already developed. We show that the PNA probe will be a candidate for a rapidly penetrating and non-degrading probe for WISH.