Mathematical analysis of involvement ratio between central and peripheral COX-2 in rat pain models with two types of COX-2 inhibitors with different distribution, celecoxib and CIAA.

The purpose of this study is to clarify involvement ratios between central and peripheral cyclooxygenase (COX)-2 in rat inflammatory pain models, by evaluating celecoxib and [6-chloro-2-(4-chlorobenzoyl)-1H-indol-3-yl]acetic acid (CIAA) on carrageenan-induced mechanical and thermal hyperalgesia. Celecoxib and CIAA exhibited ID(30) values with 1.5 and 7.7 mg/kg on mechanical hyperalgesia, respectively, and ID(25) values with 0.54 and 36 mg/kg on thermal hyperalgesia, respectively. By solving quadratic functional analysis with prostaglandin E(2) (PGE(2)) inhibitory activities, it was calculated that involvement ratios between central and peripheral COX-2 involvement were 0.47 and 0.53 on mechanical hyperalgesia, and 0.97 and 0.03 on thermal hyperalgesia, respectively. These data suggest that central and peripheral COX-2 are equally involved in mechanical hyperalgesia, while central COX-2 is predominantly involved in thermal hyperalgesia.

Characterization of primary sensory neurons mediating static and dynamic allodynia in rat chronic constriction injury model.

Mechanical allodynia, such as static and dynamic allodynia, is a prominent feature of neuropathic pain syndromes. The aim of this study is to characterize primary sensory neurons mediating the mechanical allodynia in a rat chronic constriction injury (CCI) model with a combination of pharmacological and histological investigations. N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide (BCTC), a selective and competitive antagonist of the vanilloid receptor 1 (TRPV1), and resiniferatoxin, which causes desensitization of TRPV1-expressing fibres, suppressed static allodynia but not dynamic allodynia in CCI rats. Immunohistochemical studies of TRPV1 and NF200, an A-fibre marker 200 kDa neurofilament, in dorsal root ganglion neurons demonstrated that each 48% of the positive-stained neurons were immunoreactive only for TRPV1 or NF200. The other 4% of stained neurons were double-positive for TRPV1 and NF200. Of the TRPV1positive neurons, more than 99% were small-(diameter <25 microm) and medium-(25-45 microm) sized. In contrast, 97% of NF200 single-labelled neurons were medium-and large-(>45 microm) sized. These findings suggest that two types of mechanical allodynia are transmitted by different primary sensory neurons: static allodynia is mediated by TRPV1 positive small-and medium-sized neurons and dynamic allodynia might be signalled by TRPV1-negative medium- and large-sized neurons.

Diclofenac, a non-steroidal anti-inflammatory drug, suppresses apoptosis induced by endoplasmic reticulum stresses by inhibiting caspase signaling.

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used in the treatment of inflammation and pain. In many reports, NSAIDs have induced apoptosis in a variety of cell lines such as colon cancer cells. On the other hand, more recently a few reports have found that NSAIDs protect against apoptosis. Here we investigate endoplasmic reticulum (ER)-stress-induced apoptosis of neuronal cells. The aim of this study is to examine the involvement of NSAIDs, in particular diclofenac, on ER-stress-induced apoptosis of human neuroblastoma SH-SY5Y cells. Diclofenac significantly suppressed SH-SY5Y cell death induced by two types of ER-stress-inducing agents: thapsigargin, an inhibitor of Ca2+-ATPase on the endoplasmic reticulum membrane, and tunicamycin, a glycosylation blocker. Other NSAIDs, such as indomethacin, ibuprofen, aspirin, and ketoprofen, also suppressed ER-stress-induced SH-SY5Y cell death. The dose-dependent anti-apoptotic effect of diclofenac did not correlate with the reduction of prostaglandin release. Administration of prostaglandin E2, which was a primary product of arachidonic metabolism, showed no effects against anti-apoptotic effects produced by diclofenac. Thapsigargin and tunicamycin each significantly activated caspase-3, -9, and -2 in the intrinsic apoptotic pathway in SH-SY5Y cells. Diclofenac suppressed the activation of caspases induced by both ER stresses. Thapsigargin and tunicamycin decreased the mitochondrial membrane potential in SH-SY5Y cells. Diclofenac suppressed the mitochondrial depolarization induced by both ER stresses. Diclofenac inhibited ER-stress-induced apoptosis of SH-SY5Y cells by suppressing the activation of caspases in the intrinsic apoptotic pathway. This is the first report to find that diclofenac has protective effects against ER-stress-induced apoptosis.

CyclinB2 and BIRC5 genes as surrogate biomarkers for neurite outgrowth in SH-SY5Y subclonal cells.

Neurite outgrowth plays a key role in neuronal development and regeneration, and is the hallmark assay for the effects of neurotrophic factors such as nerve growth factor (NGF). However, measuring neurite outgrowth is a slow and resource-intensive process. We therefore wanted to identify surrogate biomarkers for neurite outgrowth activity by gene expression analysis in SH-O10 cells, a subclone of the human SH-SY5Y neuroblastoma cell line but with much higher NGF-induced neurite outgrowth activity. Microarray analysis identified seven genes where mRNA levels were changed. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, were confirmed by quantitative real-time RT-PCR. Levels of NGF-induced decreases in CyclinB2 and BIRC5 mRNA in several SH-SY5Y subclones with different neurite outgrowth responses correlated with their neurite outgrowth activities. Decreases in CyclinB2 and BIRC5 mRNA induced by FK506 or retinoic acid, both of which exert potentiation of NGF-induced neurite outgrowth effects but with different mechanisms, also correlated with their neurite outgrowth activities. In conclusion, decreasing levels of CyclinB2 and BIRC5 mRNA strongly correlate with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Y subclonal cells, and have potential to become quantitative surrogate biomarkers for measuring NGF-related neurite outgrowth.

Hydroxyimine NO-donors; FK409 and derivatives.

FK409 was discovered during a screening program for prostaglandin like compounds from microbial products by measuring inhibition of platelet aggregation and vasodilation. FK409, a semisynthetic compound derived from acidic nitrosation of microbial broth, was shown to be a potent vasodilator with a similar pharmacological profile to organic nitrates such as nitroglycerin (NTG). FK409 dilated the larger diameter coronary vascular vessels more potently than those with a smaller diameter in vitro, and was more potent than NTG in a dog angina pectoris model. Clinical development of FK409 for angina pectoris included a preliminary open efficacy study in about twenty patients with angina pectoris showing an improvement in 60-70 % of patients. Anemia proved a drug related adverse event. A new study was carried out on around 20 patients with ischemic heart disease, but in the longer term the anemia remained. It was concluded that FK409 had a comparable efficacy to organic nitrates, but an undesirable adverse effect, and development was terminated. Back-up compounds for FK409, explored improvement of the pharmacokinetic profile: an increase in duration of action and a reduction of the risk of anemia. The pharmacological action of FK409 was associated with increased cGMP levels in aortic smooth muscle; and release of NO was observed by physicochemical methods. Synthesis of chemically more stable derivatives of FK409 with slower NO release was aimed at longer pharmacokinetic profiles and a lower incidence of anemia. FR144220 and FR146881 were identified as chemically more stable compounds with a longer duration of pharmacological action.

Protective effect of FK506 against apoptosis of SH-SY5Y cells correlates with regulation of the serum inducible kinase gene.

Recently, we established an in vitro model of apoptosis induced by exposure of neuroblastoma SH-SY5Y cells to thapsigargin, an endoplasmic reticular calcium-ATPase inhibitor, and demonstrated that FK506 (tacrolimus) protected against apoptosis. The purpose of this paper was to investigate a possible correlation between the protective effect of FK506 against apoptosis and the regulation of the serum inducible kinase (SNK) and fibroblast growth factor inducible kinase (FNK) genes-which are polo-like kinases expressed abundantly in the brain by FK506. Thapsigargin increased the mRNA level of SNK and FNK in SH-SY5Y cells. FK506 inhibited the increase in SNK mRNA but not FNK mRNA. Deletion analysis of the SNK promoter showed that the promoter site, which was regulated by thapsigargin and FK506 in a calcineurin-dependent manner, is a cAMP response element (CRE)/activating transcription factor (ATF)-like element located 84 base pairs (bp) proximal to the transcriptional initiation site. Although transcription of the SNK gene was also regulated by tunicamycin, etoposide, or staurosporine, FK506 did not show any effects on these regulations. We recently reported that FK506 did not protect against apoptosis induced by these agents. These results indicate that the induction of SNK mRNA by thapsigargin in SH-SY5Y cells is regulated by FK506 via an inhibition of calcineurin at the transcriptional stage, and the transcriptional regulation of the SNK gene by FK506 was well correlated with the protective effect of the compound against apoptosis. Thus, transcriptional regulation of the SNK gene may be a biological marker for analysis of apoptosis of SH-SY5Y cells.

Quantitative analysis of cadherin-11 and ß-catenin signalling during proliferation of rheumatoid arthritis-derived synovial fibroblast cells.

OBJECTIVES: Cadherin-11 (CDH11) is an adhesion molecule that anchors ß-catenin and is involved with various functions of synovial fibroblast cells (SFCs) during the development of rheumatoid arthritis (RA). However, the mechanism of CDH11 during RA-SFC proliferation is unclear. The aim of our study was to clarify the involvement of CDH11 and ß-catenin signalling during proliferation. METHODS: IL-1ß-induced and tumour necrosis factor-α (TNF-α)-induced cell proliferation, with CDH11 siRNAs, ß-catenin-specific siRNAs and a CDH11-neutralizing antibody, were assessed by 5-Bromo-2'-deoxy-uridine ELISA. KEY FINDINGS: Using CDH11 siRNAs, there were a 42% reduction in IL-1ß-induced proliferation and a 64% reduction in ß-catenin protein. When ß-catenin siRNAs were applied, there was a 63% reduction in IL-1ß-induced proliferation. The median effective concentration (EC50 ) values for IL-1ß-induced proliferation via CDH11-mediated ß-catenin-dependent, total ß-catenin-dependent and ß-catenin-independent signalling were 0.0015, 0.016 and 0.18 ng/ml, respectively. Blocking CDH11 ligation with a CDH11-neutralizing antibody did not decrease IL-1ß-induced proliferation. CONCLUSIONS: CDH11-mediated ß-catenin signalling was 42% involved in IL-1ß-induced proliferation and had the highest susceptibility to IL-1ß among the proliferative signallings analysed in this study. The mode of action for CDH11 during the cell proliferation was likely associated with a pool of ß-catenin protein. In contrast, CDH11 and ß-catenin were not involved in TNF-α-induced RA-SFC proliferation.

Implications of circadian gene expression in kidney, liver and the effects of fasting on pharmacogenomic studies.

Pharmacogenomics offers the potential to define metabolic pathways and to provide increased knowledge of drug actions. We studied relative levels of gene expression in the rat using a microarray with 8448 rat UniGenes (1928 known genes, 6520 unknown ESTs) in the liver and kidney as a function of time of day and then of feeding regime, which are common variables in preclinical pharmacogenomic studies. We identified 597 genes, including several key metabolic pathways, whose relative expression levels are significantly affected by time of day: expression of some was further modified by feeding state. These would have sparked interest in a pharmacogenomic study. Our study demonstrates that two common variables in pharmacogenomic studies can have dramatic effects on gene expression. This study provides investigators with baseline information for both kidney and liver with respect to 'normal' changes in gene expression influenced by time of day and feeding state. It also identifies 18 new genes that should be investigated for a role in circadian rhythms in peripheral tissues.

Detailed in vitro pharmacological analysis of FK506-induced neuroprotection.

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.

Suppressive effects of FR167653, an inhibitor of p38 mitogen-activated kinase, on calreticulin mRNA expression induced by endoplasmic reticulum stresses.

Several endoplasmic reticulum chaperones are simultaneously transactivated in response to various forms of endoplasmic reticulum stresses. Calreticulin is one such chaperone. We here show that the compound FR167653 [1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate] suppresses the transactivation of calreticulin following endoplasmic reticulum stress. FR167653, like SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole], has been reported to inhibit p38 mitogen-activated kinase (p38 MAPK). In this study, FR167653 concentration-dependently inhibited the up-regulation of the calreticulin mRNA level following an endoplasmic reticulum stress induced by thapsigargin in human embryonic kidney 293 (HEK293) cells and rat phechromocytoma PC12 cells. The compound concentration-dependently suppressed the transactivation of luciferase by thapsigargin in a reporter assay with a calreticulin promoter-luciferase conjugated reporter vector. SB203580 also significantly suppressed the transactivation of calreticulin by thapsigargin. Therefore, FR167653 regulated the mRNA expression of calreticulin at the transcriptional level without affecting the stability of the mRNA, as well as via inhibition of p38 MAPK activated by thapsigargin. FR167653 also inhibited the transactivation of calreticulin stimulated by two other endoplasmic reticulum stress inducers, tunicamycin and A23187. Moreover, the inhibitory action of the compound on the transactivation was observed in other cell lines. The calreticulin promoter region includes three sequential cis-acting endoplasmic reticulum stress response elements (ERSEs). As each of these ERSEs was sequentially deleted, there was an increasing loss of the transactivation by thapsigargin or tunicamycin. FR167653 inhibited the transactivation in all the reporter plasmid constructs containing the calreticulin promoter region with an ERSE/ERSEs. In conclusion, FR167653 is the first compound shown to inhibit the transactivation of calreticulin following various endoplasmic reticulum stresses. The suppressive effects of the compound were considered to be due to an inhibition of the signaling leading to ERSEs activation in the calreticulin promoter region via an inhibition of p38 MAPK, which is activated by endoplasmic reticulum stresses.

Inhibition of arterial thrombosis by a protease-activated receptor 1 antagonist, FR171113, in the guinea pig.

The antiplatelet and antithrombotic effects of FR171113, 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one, a non-peptide protease-activated receptor 1 (PAR1) antagonist, were evaluated in guinea pigs. FR171113 inhibited Ser-Phe-Leu-Leu-Arg-Asn-NH2 (a synthetic PAR1 agonist peptide)-induced and thrombin-induced aggregation of guinea pig platelets in a concentration-dependent manner in vitro (IC50=1.5 and 0.35 microM, respectively). Subcutaneous administration of FR171113 (0.1-3.2 mg/kg) produced a dose-dependent inhibition of platelet aggregation ex vivo. The ED50 value of FR171113 for platelet aggregation was 0.49 mg/kg s.c. However, FR171113 did not have an inhibitory effect on ADP- or collagen-induced platelet aggregation in vitro and ex vivo. One hour after FR171113 treatment at 1.0 mg/kg s.c., significant inhibition of arterial thrombosis without a prolongation of thrombin time or coagulation time was seen in the FeCl3-induced carotid artery thrombosis model in guinea pigs. Furthermore, FR171113 did not prolong bleeding time even at 32 mg/kg s.c., which is a much higher dose than that required in the thrombosis model. These observations indicate that FR171113 has desirable antiplatelet effects both in vitro and in vivo and that its in vivo antithrombotic activity is efficacious without causing a prolongation of bleeding time.

Discovery of novel inhibitors of inducible nitric oxide synthase.

We have discovered three compounds, 5-chloro-1,3-dihydro-2H-benzimidazol-2-one (FR038251), 1,3(2H,4H)-isoquinolinedione (FR038470) and 5-chloro-2,4(1H,3H)-quinazolonedione (FR191863), which show inhibition of inducible nitric oxide synthase (iNOS). The iNOS inhibitory activity of the compounds was examined in comparison with that of aminoguanidine, a representative iNOS inhibitor. FR038251, FR038470 and FR191863 inhibited mouse iNOS, with IC50 values of 1.7, 8.8 and 1.9 microM, respectively, in an in-vitro enzyme assay. These inhibitory activities are comparable with that of aminoguanidine (IC50 = 2.1 microM). The three compounds had iNOS selectivity 38- and 8-times, > 11- and 3-times, and 53- and 3-times compared with rat neuronal NOS and bovine endothelial NOS, respectively. These compounds concentration-dependently inhibited NO production in intact RAW264.7 mouse macrophages stimulated by lipopolysaccharide (LPS)/interferon-gamma. At 100 microM, FR038251, FR038470, FR191863 and aminoguanidine showed 81, 44, 54 and 78% inhibition of NO production, respectively. In an in-vivo experiment, FR038251, FR038470, FR191863 and aminoguanidine inhibited NO production in LPS-treated mice by 68, 40, 5 and 68% at an oral dose of 100 mg kg-1. The in-vivo inhibitory activity of FR038251 was almost identical to that of aminoguanidine. In conclusion, the three FR compounds are iNOS inhibitors with novel structures and may be candidate compounds leading to discovery of more iNOS inhibitors in the future.

Involvement of inflammation in severe post-operative pain demonstrated by pre-surgical and post-surgical treatment with piroxicam and ketorolac.

OBJECTIVES: Post-operative pain is considered to involve inflammation caused by tissue injury. However, the mechanism and timing of the involvement of inflammation in the post-operative pain remain complicated because they can vary among different types of surgery. In this study a rat incision model was used to investigate how inflammation induced by cyclooxygenases (COXs) is involved in severe post-operative pain. METHODS: Longitudinal incision with a length of 1cm was made through skin and fascia on the right hind paw of rats, starting 0.5cm from the edge of the heel and extending towards the toes. Allodynia was evaluated using the von Frey hair test and its degree was recorded as the paw withdrawal threshold (PWT). Two non-steroidal anti-inflammatory drugs (NSAIDs), piroxicam and ketorolac, were given to rats after or before surgery, and the effects of the drugs on allodynia induced by the hind paw incision were examined. KEY FINDINGS: The PWT reduction reached a sub-maximal level at 3h, a maximal level at one day after the surgery and lasted for more than 8 days, with the parallel development of inflammation (characterized by cell infiltration and oedema). Treatment with morphine (1mg/kg, s.c.) at one day after the surgery showed a significant anti-allodynic effect. Treatment with either piroxicam (10mg/kg, p.o.) or ketorolac (10mg/kg, p.o.) at one day after the surgery did not exhibit significant anti-allodynic effect, whereas pre-surgical treatment with each drug showed significant anti-allodynic effects at 3h after surgery. CONCLUSIONS: These findings suggest the involvement of cyclooxygenases in evoking pain that occurs in the immediate post-operative period, and that an initial suppression of rapid inflammation by treatment with NSAIDs before major surgery plays an important role in the management of severe post-operative pain.

Discovery of a novel neuroprotective compound, AS1219164, by high-throughput chemical screening of a newly identified apoptotic gene marker.

We have reported that tacrolimus (FK506), an immunosuppressive drug, and diclofenac, a non-steroidal anti-inflammatory drug, possess different modes of neuroprotective action. FK506 suppresses only thapsigargin-induced apoptosis in neuroblastoma SH-SY5Y cells while diclofenac reverses tunicamycin-induced as well as thapsigargin-induced apoptosis. The aim of this study is to discover novel compounds that exert neuroprotective properties by using the transcriptional response of a newly identified gene, which was regulated by both FK506 and diclofenac, as a surrogate screening marker in high-throughput chemical screening and characterize the compounds in comparison with FK506 and diclofenac. Using a microarray with 4504 human cDNAs and quantitative RT-PCR, two genes as apoptotic markers, transmembrane protein 100 (TMEM100) and limb-bud and heart (LBH), were identified because the thapsigargin-induced elevations in their mRNA levels were reversed by both FK506 and diclofenac. A luciferase reporter assay with a TMEM100 promoter region was applied to high-throughput chemical screening. AS1219164, {3-[(E)-2-{5-[(E)-2-pyridin-4-ylvinyl]pyridin-3-yl} vinyl]aniline}, suppressed thapsigargin-induced transactivation of the TMEM100 gene and reversed thapsigargin-induced increases in TMEM100 and LBH mRNA levels in SH-SY5Y cells, similar to the effects of FK506 and diclofenac. Furthermore, AS1219164 protected against SH-SY5Y cell death induced by four apoptotic agents including thapsigargin, similar to diclofenac, but was more potent than diclofenac, while FK506 only showed protective effects against thapsigargin-induced cell death. In conclusion, a novel neuroprotecitve compound, AS1219164, was discovered by high-throughput chemical screening using a reporter assay with the TMEM100 gene promoter regulated by both FK506 and diclofenac. Reporter assay using the promoter region of a gene under pharmacological and physiological transcriptional regulation would be well suit for use in high-throughput chemical screening.

Pharmacologic investigation of the mechanism underlying cold allodynia using a new cold plate procedure in rats with chronic constriction injuries.

Cold allodynia is a frequent clinical symptom of patients with neuropathic pain. Despite numerous studies of cold allodynia, using animal models of neuropathic pain, little is known about its underlying mechanisms. This study was performed to establish a method for the pharmacologic evaluation of cold allodynia using several analgesics in a chronic constriction injury (CCI) rat model of neuropathic pain. Compared with the results obtained before the CCI operation, the CCI rats placed on a cork plate at 20 degrees C exhibited a slight change in the paw withdrawal latency because of the mechanical stimulus mediated by the injured paw touching the plate. By contrast, there was a significant reduction in the paw withdrawal latency on a cold metal plate compared with that on the cork plate after the CCI surgery, with the maximum decrease occurring on postoperative day 7. This reduction is thought to specifically reflect cold-induced pain behavior. In addition, both naïve and CCI rats showed behavioral changes at 5 and 0 degrees C, but not at 10 degrees C or higher. Interestingly, a subcutaneous morphine dose of 6 mg/kg completely inhibited cold allodynia induced at 10 degrees C on postoperative day 7. Under this condition, both the sodium channel blocker mexiletine (10 and 30 mg/kg, subcutaneously) and the calcium channel alpha2delta subunit blocker pregabalin (30 and 100 mg/kg, orally) significantly suppressed cold allodynia. Additionally, both resiniferatoxin (0.3 mg/kg, subcutaneously), an ultrapotent analog of capsaicin that desensitizes C fibers, and the VR1 channel antagonist N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (10 and 30 mg/kg, orally) significantly prolonged the paw withdrawal latency. In conclusion, our data suggest that the activation of C fibers mediates cold allodynia.

Neuroprotective efficacy of the peroxisome proliferator-activated receptor delta-selective agonists in vitro and in vivo.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as ligand-modulated transcription factors that regulate gene expression in many important biological processes. The PPARdelta subtype has the highest expression in the brain and is postulated to play a major role in neuronal cell function; however, the precise physiological roles of this receptor remain to be elucidated. Herein, we show that the high-affinity PPARdelta agonists L-165041 [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid] and GW501516 [2-methyl4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid] protect against cytotoxin-induced SH-SY5Y cell injury in vitro and both ischemic brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo. In the SH-SY5Y studies, treatment with L-165041 or GW501516 significantly and concentration-dependently attenuated cell death following thapsigargin, 1-methyl-4-phenylpyridinium, or staurosporine exposure, with the extent of damage correlated with the level of caspase-3 inhibition. In the transient (90 min) middle cerebral artery occlusion model of ischemic brain injury in rats, i.c.v. infusion of L-165041 or GW501516 significantly attenuated the ischemic brain damage measured 24 h after reperfusion. Moreover, the PPARdelta agonists also significantly attenuated MPTP-induced depletion of striatal dopamine and related metabolite contents in mouse brain. These results demonstrate that subtype-selective PPARdelta agonists possess antiapoptotic properties in vitro, which may underlie their potential neuroprotective potential in in vivo experimental models of cerebral ischemia and Parkinson's disease (PD). These findings suggest that PPARdelta agonists could be useful tools for understanding the role of PPARdelta in other neurodegenerative disorders, as well as attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.

An epoch-making application of discharge plasma phenomenon to gene-transfer.

We discovered an epoch-making gene transfer method utilizing discharge plasma. Although an electroporation method is commonly used in present gene transfer experiments, it cannot transfer genes into primary cells sufficiently. The atmospheric pressure discharge plasma employed in this study was originally used for surface treatment of non-biological materials. We hypothesized that it could provide a suitable effect on the surface of target cells and applied it to gene transfer into various types of cells. The plasma technology succeeded in the efficient transfer of green fluorescence protein (GFP) plasmid into post-mitotic neuronal cells obtained from cerebral cortices of rats, into which an electroporation with conventional equipment cannot transfer genes sufficiently, as the cells were attached. After the transfection of rat pheochromocytoma PC12 cells with the GFP gene by plasma treatment, the cells retained their function, that is, nerve growth factor-induced differentiation. Furthermore, gene transfer with the plasma technology was also applicable to other types of cell lines such as HeLa cells and Chinese hamster lung (CHL) cells as adherent cell lines, and Jurkat cells as a suspended cell line, and another type of primary cell, human umbilical vein endothelial cells (HUVEC). In conclusion, the plasma method is an epoch-making gene transfer technology which efficiently transfers genes into primary cells into which electroporation cannot transfer genes. Moreover, the method is able to universally transfer genes into various types of cells as the function of the cells was maintained.

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor.

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.

Mass-tag technology for monitoring of protein kinase activity using mass spectrometry.

Monitoring of intracellular protein kinase activity is very important for fields involving diagnosis and drug screening. However, current methods, such as radiometry using (32)P, or ELISA, are laborious and time-consuming. We have developed high-throughput assay system of protein kinase activity using mass-tagged substrate peptide probes and mass spectrometry. This assay system can easily evaluate target kinase activity and will potentially be able to simultaneously profile many protein kinase activities.

Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation.

The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1- and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1- and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5' regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.