The Wakan-yaku Universe: A Useful Authorized Traditional Concept for Developing Novel Therapeutic Categories and Medicinal Drugs.

This review article mentions about the following points, and proposes its importance and positive thinking. 1) Wakan-yaku (Japanese oriental medicines) is covered by the national health insurance system in Japan as therapeutic drugs to be actively used in medical practice to treat illness. 2) Applications of Wakan-yaku is accomplished based on the reliable own theories which are established with long histories. 3) Promotion of studies based on these theories will be highly expected to find novel view points which breaks conventional concepts and to novel standards for developing new medicinal drugs. Although studies based on the reliable Wakan-yaku theories are not advancing satisfactorily till now, the possibilities to obtain the advanced resources for drugs and novel viewpoints for experiments by studies about Wakan-yaku theories are discussed in this review.

Comparing the effects of kamikihito in Japan and kami-guibi-tang in Korea on memory enhancement: working towards the development of a global study.

Traditional medicine is widely used in East Asia, and studies that demonstrate its usefulness have recently become more common. However, formulation-based studies are not globally understood because these studies are country-specific. There are many types of formulations that have been introduced to Japan and Korea from China. Establishing whether a same-origin formulation has equivalent effects in other countries is important for the development of studies that span multiple countries. The present study compared the effects of same-origin traditional medicine used in Japan and Korea in an in vivo experiment. We prepared drugs that had the same origin and the same components. The drugs are called kamikihito (KKT) in Japan and kami-guibi-tang (KGT) in Korea. KKT (500 mg extract/kg/day) and KGT (500 mg extract/kg/day) were administered to ddY mice, and object recognition and location memory tests were performed. KKT and KGT administration yielded equivalent normal memory enhancement effects. 3D-HPLC showed similar, but not identical, patterns of the detected compounds between KKT and KGT. This comparative research approach enables future global clinical studies of traditional medicine to be conducted through the use of the formulations prescribed in each country.

Serotonin 2C receptor as a superhero: diversities and talents in the RNA universe for editing, variant, small RNA and other expected functional RNAs.

The serotonin 2C receptor subtype (5-HT2C) has a unique profession and continues to provide exciting and critical new information. The 5-HT2C is modulated at the RNA level by several mechanisms, including editing, short variant generation, and small RNAs. Recently, these phenomena, which had been demonstrated individually, were shown to be associated with each other. At present, many reports provide information about the influence of RNA regulation on receptor protein activities and expression, which was thought to be the final functional product. However, complicated behavior at the RNA stage allows us to imagine that the RNA itself has functional roles in the RNA universe. The 5-HT2C RNA may play several roles. This review will outline previous 5-HT2C studies and prospects for future studies.

Clarifying the pharmacological mechanisms of action of Shenfu Decoction on cardiovascular diseases using a network pharmacology approach.

Since the molecular mechanisms underlying in the pathogenesis of cardiovascular diseases (CVD) are extremely complex and have not yet been elucidated in detail, CVD remain the leading cause of death worldwide. Traditional Chinese medicine involves the treatment of disease from an overall perspective, and its therapeutic effects on CVD have been demonstrated. However, the mechanisms contributing to the multiscale treatment of cardiovascular diseases at the systematic level remain unclear. Network pharmacology methods and a gene chip data analysis were integrated and applied in the present study, which was conducted to investigate the potential target genes and related pathways of Shenfu Decoction (SFD) for the treatment of myocardial injury. The gene chip analysis was initially performed, followed by network pharmacology to identify differentially expressed genes (DEG) and a functional enrichment analysis. Protein-protein networks were constructed and a module analysis was conducted. A network analysis was used to identify the target genes of SFD. Regarding the results obtained, 1134 DEG were identified using the STRING website. The module analysis revealed that nine hub genes exhibited ubiquitin-protein ligase activity. Therefore, SFD significantly alters the expression of ubiquitination-related genes and, thus, plays an important therapeutic role in the treatment of heart failure. In conclusion, hub genes may provide a more detailed understanding of the molecular mechanisms of action of as well as candidate targets for SFD therapy.

Use of an electrophysiological technique for stepwise detection of trace agonist constituents of Hochuekkito in Xenopus oocytes injected with serotonin 2C receptor mRNA.

An electrophysiological bioassay was used to isolate the active compound from Hochuekkito (HET), which the current authors previously described as having potent agonist action against serotonin 2C receptors (5-HT2CR). Synthetic 5-HT2CR mRNA was injected into Xenopus oocytes to specifically express these receptors. Crude extracts and purified products were subjected to an electrophysiological bioassay using the voltage clamp method. HET stimulated a 5-HT2CR-induced current response, whereas Juzentaohoto (JTT), which has anti-depressive action similar to that of HET, did not. Current responses were not observed with an extract mixed with five types of herbal medicines common to HET and JTT but were detected with an extract with the five types of herbal medicines found in HET alone (Hoc5). When the responses to each of the five types of Hoc5 were examined, current responses were noted with Cimicifugae rhizoma (CR) and Citrus unshiu Markovich extracts. Since efficacy and the EC50 value were higher for CR, its constituents were separated using three-dimensional high-performance liquid chromatography and the current response at each of the isolated peaks was examined. One constituent displayed a strong response and was identified as a single substance with a molecular weight of 283.1393 based on liquid chromatography/mass spectrometry. These results will contribute to the isolation of 5-HT2CR-stimulating constituents in HET and the identification of trace constituents with agonist action.

Serotonin 2C receptor (5-HT2CR) mRNA editing-induced down-regulation of 5-HT2CR function in Xenopus oocytes: the significance of site C editing.

Serotonin 2C receptor (5-HT2CR) mRNA receives editing at 5 nucleotide positions (sites A-E) located in the sequence encoding the second intracellular loop of 5-HT2CR. 5-HT2CR mRNA without editing and with editing at sites AB, ABD, ABC, ABCD, and C are translated to 6 isoforms of 5-HT2CR: INI(non-edited), VNI(AB), VNV(ABD), VSI(ABC), VSV(ABCD), and ISI(C), respectively. In this study, we investigated electrophysiologically the ability of these isoforms to couple with the G protein/phospholipase C (PLC) system using Xenopus oocytes injected with edited 5-HT2CR RNAs and muscarinic M(1) receptor (M1R) RNA. The efficacy with which 5-HT stimulated each isoform was calculated by comparing 5-HT-induced current with 100 microM acetylcholine-induced M1R current. Stimulation with 5-HT of INI(non-edited), VNI(AB), VNV(ABD), VSI(ABC), VSV(ABCD), and ISI(C) expressed in Xenopus oocytes showed concentration-dependent responses with EC(50) values of 8.6, 17.2, 76,5, 22.0, 91.2, and 20.3 nM, respectively. No significant difference in the ability of 5-HT to induce currents among the oocytes expressing these isoforms was detected, but in the oocytes expressing VSI(ABC) or VSV(ABCD), 5-HT had a significantly reduced ability to induce currents. These results suggest that editing at site C together with sites A and B and/or D markedly reduces 5-HT2CR function by generating isoforms with reduced ability to activate PLC.

Possible involvement of signal transducers and activators of transcription 3 system on depression in the model mice brain.

The aim of this study was to examine gene expression changes in the frontal cortex and hippocampus of animals with different susceptibility to stressful stimuli. Using a learned helplessness (LH) paradigm, mice received moderate current stimulation to induce different extents of LH behavior. Changes in mRNA expression were investigated by microarray and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Comparisons of expression profiles between LH and control or non-learned helplessness (non-LH) animals revealed that the signal transducers and activators of transcription 3 (Stat3)-interacting protein (StIP1) gene is downregulated in LH animals and the suppressor of cytokine signaling 3 (Socs3) gene is up-regulated in non-LH animals. Since both StIP1 and Socs3 regulate the activity of Stat3 gene, these results suggest that Stat3 systems may be involved in the pathogenesis of depressive disorders.

Enhanced expression of BCL2/adenovirus EIB 19-kDa-interacting protein 3 mRNA, a candidate for intrinsic depression-related factor, and effects of imipramine in the frontal cortex of stressed mice.

We previously reported that long-term treatment with some antidepressants at low concentrations upregulates BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) mRNA expression in NG108-15 cells without causing cell damage, suggesting that BNIP3 is a candidate of intrinsic depressive disorder-related factor(s). In this study, to clarify the physiologic functions of BNIP3, we investigated whether BNIP3 is actually related to the depressive condition in the brain using learned helplessness (LH) mice, an animal model of depression. Based on the score of escape failure, an index of depression degree, stressed animals were divided into groups with LH and without depressive-like symptoms (i.e., non-depressed phenotype, non-LH). The score of escape failure of the LH group was decreased after 14 d of treatment with imipramine in a dose-dependent manner. BNIP3 mRNA expression was enhanced in both the LH and non-LH groups. Imipramine treatment at 5 and 20 mg/kg/d enhanced BNIP3 mRNA expression only in the LH group but not in non-LH group or non-stressed group. These results raise the possibility that BNIP3 acts as an antistress factor in the brain.

Influence of an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, on 5-HT2CR mRNA editing in primary cultured cortical cells.

Treatment of primary cultured cortical cells with erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), an inhibitor of adenosine deaminase (ADAR), for 6 d significantly and concentration-dependently reduced the editing efficacy at sites C and D but not at site A or B of 5-HT2CR mRNA. The treatment failed to affect the editing of ADAR-2 pre-mRNA and a subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor (GluR2) mRNA. These findings suggest that EHNA is useful for clarifying the functional roles of 5-HT2CR mRNA editing at sites C and D.

BNIP-3: a novel candidate for an intrinsic depression-related factor found in NG108-15 cells treated with Hochu-ekki-to, a traditional oriental medicine, or typical antidepressants.

Wakan-yaku is a type of Japanese and Sino traditional, systematized medical care that has been practiced for hundreds of years. To search for novel intrinsic factors related to the action of antidepressants, we used Hochu-ekki-to (HET), a Wakan-yaku medicine with antidepressive effects. First, we verified the quality of the HET by three-dimensional high-performance liquid chromatography and a cytotoxicity check in NG108-15 cells. We performed a DNA microarray analysis of the gene expression in cells treated with 50 micro/ml HET for more than 20 days. HET enhanced the expression of 125 (2.9%) genes and decreased the expression of 255 (6.0%) genes among the 4277 genes that were tested. The concentration-dependent increase in the expression of BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP-3) mRNA was particularly remarkable. A concentration-dependent increase in the expression of BNIP-3 mRNA was also observed when cells were treated with imipramine, mianserin, or milnacipran. These results suggest that BNIP-3 is a candidate for an intrinsic factor related to antidepressive effects and that Wakan-yaku theory may be useful for the identification of other intrinsic functional molecules.

Developmental changes in expression and self-editing of adenosine deaminase type 2 pre-mRNA and mRNA in rat brain and cultured cortical neurons.

Adenosine deaminase-1 and -2 (ADAR-1 and -2) are double-stranded RNA-specific enzymes involved in the editing of genes including serotonin 2C receptor (5-HT2CR) mRNA and ADAR-2 pre-mRNA. We reported that the editing efficacy of 5-HT2CR mRNA altered during brain development in rats. The present study aimed to clarify if changes in the expression of ADAR genes and the editing of ADAR-2 pre-mRNA occur during development. The expression level of ADAR-1 mRNA was constant during development, whereas the expression levels of ADAR-2 mRNA and ADAR-2 pre-mRNA markedly increased during development. ADAR-2 pre-mRNA possesses six editing sites. Editing of these sites did not occur during the embryonic period; however, the number of edited sites and the editing frequency at these sites increased after birth and cultivation period. These results suggest that the increases in ADAR-2 pre-mRNA editing and mRNA expression of the enzyme may play a role in development. We also discuss the relationship between 5-HT2CR mRNA editing and the expression/RNA editing of ADAR-1 and ADAR-2 mRNA.

Extracellular cathepsin L stimulates axonal growth in neurons.

OBJECTIVE: Cathepsin L, a lysosomal endopeptidase expressed in most eukaryotic cells, is a member of the papain-like family of cysteine proteases. Although commonly recognized as a lysosomal protease, cathepsin L is also secreted and involved in the degradation of extracellular matrix proteins. Previous studies demonstrated that the secretion of cathepsin L was stimulated by basic fibroblast growth factor (bFGF) and bFGF-enhanced axonal terminal sprouting of motor neurons. Based on these results, although it has never been directly investigated, we hypothesized that extracellular cathepsin L may induce axonal growth. RESULTS: To confirm the hypothesis, the axonal growth activity of recombinant cathepsin L was evaluated in cultured cortical and spinal cord neurons. Treatment with recombinant cathepsin L significantly enhanced axonal growth, but not dendritic growth. This result indicated that extracellular cathepsin L may act as a new neuronal network modulator.

A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action.

N(alpha)-vanillyl-N(omega)-nitroarginine (N - 1) that combines the active functions of natural antioxidant and nitric oxide synthase inhibitor was developed for its neuroprotective properties. N - 1 exhibited protective effects against hydrogen peroxide-induced cell damage and the inhibitory effect on nitric oxide 'NO' production induced by calcium ionophore in NG 108-15 cells. N - 1 inhibited the constitutive NOS isolated from rat cerebellar in a greater extent than constitutive NOS from human endothelial cells. Low binding energy (-10.2 kcal/mol) obtained from docking N - 1 to nNOS supported the additional mode of action of N - 1 as an nNOS inhibitor. The in vivo neuroprotective effect on kainic acid-induced nitric oxide production and neuronal cell death in rat brain was investigated via microdialysis. Rats were injected intra-peritonially with N - 1 at 75 micromol/kg before kainic acid injection (10 mg/kg). The significant suppression effect on kainic acid-induced NO and significant increase in surviving cells were observed in the hippocampus at 40 min after the induction.

Prevention of kainic acid-induced changes in nitric oxide level and neuronal cell damage in the rat hippocampus by manganese complexes of curcumin and diacetylcurcumin.

Curcumin is a natural antioxidant isolated from the medicinal plant Curcuma longa Linn. We previously reported that manganese complexes of curcumin (Cp-Mn) and diacetylcurcumin (DiAc-Cp-Mn) exhibited potent superoxide dismutase (SOD)-like activity in an in vitro assay. Nitric oxide (NO) is a free radial playing a multifaceted role in the brain and its excessive production is known to induce neurotoxicity. Here, we examined the in vivo effect of Cp-Mn and DiAc-Cp-Mn on NO levels enhanced by kainic acid (KA) and L-arginine (L-Arg) in the hippocampi of awake rats using a microdialysis technique. Injection of KA (10 mg/kg, i.p.) and L-Arg (1000 mg/kg, i.p.) significantly increased the concentration of NO and Cp-Mn and DiAc-Cp-Mn (50 mg/kg, i.p.) significantly reversed the effects of KA and L-Arg without affecting the basal NO concentration. Following KA-induced seizures, severe neuronal cell damage was observed in the CA1 and CA3 subfields of hippocampal 3 days after KA administration. Pretreatment with Cp-Mn and DiAc-Cp-Mn (50 mg/kg, i.p.) significantly attenuated KA-induced neuronal cell death in both CA1 and CA3 regions of rat hippocampus compared with vehicle control, and Cp-Mn and DiAc-Cp-Mn showed more potent neuroprotective effect than their parent compounds, curcumin and diacetylcurcumin. These results suggest that Cp-Mn and DiAc-Cp-Mn protect against KA-induced neuronal cell death by suppression of KA-induced increase in NO levels probably by their NO scavenging activity and antioxidative activity. Cp-Mn and DiAc-Cp-Mn have an advantage to be neuroprotective agents in the treatment of acute brain pathologies associated with NO-induced neurotoxicity and oxidative stress-induced neuronal damage such as epilepsy, stroke and traumatic brain injury.

Suppressive effects of isorhynchophylline on 5-HT2A receptor function in the brain: behavioural and electrophysiological studies.

Isorhynchophylline is a major oxindole alkaloid found in Uncaria species which have long been used in traditional Chinese medicine. Here, we investigated the effects of isorhynchophylline and isorhynchophylline-related alkaloids on 5-hydroxytryptamine (5-HT) receptor-mediated behavioural responses in mice and 5-HT-evoked current responses in Xenopus oocytes expressing 5-HT2A or 5-HT2C receptors. Isorhynchophylline dose-dependently inhibited 5-HT2A receptor-mediated head-twitch but not 5-HT1A receptor-mediated head-weaving responses evoked by 5-methoxy-N,N-dimethyltryptamine. Pretreatment with reserpine, a monoamine-depleting agent, enhanced the head-twitching, but did not influence the effect of isorhynchophylline on the behavioural response. Isocorynoxeine, an isorhynchophylline-related alkaloid in which the configuration of the oxindole moiety is the same as in isorhynchophylline, also reduced the head-twitch response in reserpinized mice over the same dose range as isorhynchophylline, while both rhynchophylline and corynoxeine, stereoisomers of isorhynchophylline and isocorynoxeine, did not. None of the alkaloids tested had an effect on meta-chlorophenylpiperazine-induced hypolocomotion, a 5-HT2C receptor-mediated behavioural response. In experiments in vitro, isorhynchophylline and isocorynoxeine dose-dependently and competitively inhibited 5-HT-evoked currents in Xenopus oocytes expressing 5-HT2A receptors, but had less of a suppressive effect on those in oocytes expressing 5-HT2C receptors. These results indicate that isorhynchophylline and isocorynoxeine preferentially suppress 5-HT2A receptor function in the brain probably via a competitive antagonism at 5-HT2A receptor sites and that the configuration of the oxindole moiety of isorhynchophylline is essential for their antagonistic activity at the 5-HT2A receptor.

Choto-san, a Kampo formula, improves chronic cerebral hypoperfusion-induced spatial learning deficit via stimulation of muscarinic M1 receptor.

A recent double-blind and placebo-controlled study demonstrated a beneficial effect of Choto-san, a Kampo (traditional medicine of Japan) formula, on cognitive impairment in patients with vascular dementia. However, the neuronal mechanism underlying the therapeutic effects of this formula remains to be clarified. Using a chronic cerebral hypoperfusion model, we investigated the effect of Choto-san on cognitive dysfunction in mice to clarify its mechanism of actions. Chronic cerebral hypoperfusion was induced by permanent occlusion of both the common carotid arteries (2VO). Choto-san and Uncaria, a major constituent of Choto-san, caused an improvement in 2VO-induced learning deficits, whereas Uncaria-free Choto-san did not. The effects of Choto-san and Uncaria were blocked by pirenzepine, a selective muscarinic M1 antagonist. In a tube-dominance test, 2VO induced increased rates of assertive behavior in mice. 2VO mice administered Choto-san showed significantly reduced rates of assertive behavior compared to vehicle-treated controls, whereas Uncaria-free Choto-san and Uncaria had little effect on 2VO-induced assertive behavior. 2VO caused a significant decrease in the level of acetylcholine (ACh) contents in the brain, and the daily administration of Choto-san or Uncaria raised the ACh level to that in the sham-operated controls. These results suggest that Choto-san has an ameliorating effect on the spatial memory deficit caused by chronic hypoperfusion, and that the effect is mainly attributable to Uncaria. Moreover, it was suggested that the effects of Choto-san and Uncaria are at least partly mediated by stimulation of the muscarinic M1 receptor.

Manganese complexes of curcumin and its derivatives: evaluation for the radical scavenging ability and neuroprotective activity.

In this study, three manganese complexes of curcumin (Cp) and related compounds, diacetylcurcumin (AcylCp) and ethylenediamine derivative (CpED), were synthesized and evaluated in vitro for antilipid peroxidation and superoxide dismutase activity. The manganese complexes exhibited a great capacity to protect brain lipids against peroxidation with IC50 of 6.3-26.3 microM. All manganese complexes showed much greater SOD activity than their corresponding antioxidant ligands as well as trolox with IC50 values of 8.9-29.9 microM. AcylCp and curcumin manganese complexes (AcylCpCpx and CpCpx) also gave the highest inhibitory activity to H2O2-induced cell damage (oxidative stress) at 0.1 microg/ml (< 0.2 microM) in NG108-15 cells, which were more potent than curcumin and related compounds. The neuropharmacological tests in mice supported the idea that the SOD mimicking complexes were able to penetrate to the brain as well as their role in the modulation of brain neurotransmitters under the aberrant conditions. The complexes significantly improved the learning and memory impairment induced by transient ischemic/reperfusion. AcylCpCpx, CpCpx, and CpEDCpx showed significant protection at 6.25, 25, and 50 mg/kg (i.p.), respectively, whereas manganese acetate and curcumin had no effect at doses of 50 mg/kg. In addition, treatment of AcylCpCpx and curcumin significantly attenuated MPTP-induced striatal dopamine depletion in mice, which was in accordance with the increase in the density of dopaminergic neurons when compared with MPTP-treated mice. These results support the important role of manganese in importing SOD activity and consequently, the enhancement of radical scavenging activity. AcylCpCpx and CpCpx seem to be the most promising neuroprotective agents for vascular dementia.

Pteropodine and isopteropodine positively modulate the function of rat muscarinic M(1) and 5-HT(2) receptors expressed in Xenopus oocyte.

Pteropodine and isopteropodine are heteroyohimbine-type oxindole alkaloid components of Uncaria tomentosa (Willd.) DC, a Peruvian medicinal plant known as cat's claw. In this study, the effects of these alkaloids on the function of Ca(2+)-activated Cl(-) currents evoked by stimulation of G protein-coupled muscarinic M(1) acetylcholine and 5-HT(2) receptors were studied in Xenopus oocytes in which rat cortex total RNA was translated. Pteropodine and isopteropodine (1-30 microM) failed to induce membrane current by themselves. However, these alkaloids markedly enhanced the current responses evoked by both acetylcholine and 5-hydroxyhyptamine (5-HT) in a concentration-dependent and reversible manner with the maximal effects at 30 microM. Pteropodine and isopteropodine produced 2.7- and 3.3-fold increases in the acetylcholine response with EC(50) values of 9.52 and 9.92 microM, respectively, and 2.4- and 2.5-fold increases in the 5-HT response with EC(50) values of 13.5 and 14.5 microM, respectively. In contrast, in oocytes injected with total RNA from the rat cerebellum or spinal cord, neither alkaloid had an effect on the metabotropic current responses mediated by glutamate receptor(1 and 5) (mGlu(1/5)) receptors or ionotropic responses mediated by N-methyl-D-aspartate, kainic acid or glycine. Pteropodine and isopteropodine (10 microM) significantly reduced the EC(50) values of acetylcholine and 5-HT that elicited current responses, but had no effect on the maximal current responses elicited by acetylcholine and 5-HT. On the other hand, mitraphylline, a stereoisomer of pteropodine, failed to modulate acetylcholine- and 5-HT-induced responses. These results suggest that pteropodine and isopteropodine act as positive modulators of muscarinic M(1) and 5-HT(2) receptors.

Pharmacological evidence for antidementia effect of Choto-san (Gouteng-san), a traditional Kampo medicine.

To clarify the clinical efficacy of one of the traditional medicines in the treatment of patients with vascular dementia, we investigated the pharmacological activities of Choto-san in animal models. Pretreatment with Choto-san (0.75-6.0 g/kg po), a component herb, Chotoko (75-600 mg/kg po), and indole alkaloids and phenolic fractions of Chotoko prevented ischemia-induced impairment of spatial learning behaviour in water maze performance of mice. A single administration of Choto-san (0.5 to 6.0 g/kg po) or Chotoko (Uncaria genus) produced a dose-dependent antihypertensive effect in spontaneously hypertensive rats (SHR) and partly inhibited the induction of the apoplexy in stroke-prone SHR (SHR-SP). Choto-san, Chotoko, and its phenolic constituents, (-)epicatechin and caffeic acid, significantly protected NG108-15 cells from injury induced by H(2)O(2) exposure in vitro and also inhibited lipid peroxidation in the brain homogenate. Indole alkaloids, rhynchophylline and isorhynchophylline (1-100 microM), reversibly reduced N-methyl-D-aspartate (NMDA)-induced current concentration dependently in NMDA receptor-expressed Xenopus oocytes. These results suggest that antidementia effects of Choto-san are due to antihypertensive, free radical scavenging and antiexcitotoxic effects, which are attributed at least partly to phenolic compounds and indole alkaloids contained in Chotoko.

Determination method of sense sequence based on RT/PCR.

When novel sequences are isolated by differential display and other methods, it seems useful to determine which is a sense sequence at an early stage before further experiments. A novel sequence, named MT-001, which shows enhanced expression in the permanent ischemic rat brain, was isolated by differential display. Based on this sequence, a primer set for both direction was designed. Each primer was used to make a cDNA and PCRs performed with each cDNA and both primers. One primer used in the RT step produced a PCR product at the expected position, but another primer in the reverse direction could not. This result indicated that the primer that made the expected PCR product is antisense.