High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus (HTLV-1). SIRT1, a nicotinamide adenine dinucleotide(+)-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, owing to its ability to deacetylate numerous substrates, such as histone and NF-κB, which is implicated as an exacerbation factor in ATL. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines. Sirtinol-induced apoptosis was mediated by activation of the caspase family and degradation of SIRT1 in the nucleus. Furthermore, SIRT1 knockdown by SIRT1-specific small interfering RNA caused apoptosis via activation of caspase-3 and PARP in MT-2 cells, HTLV-1-related cell line. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.

Target epitopes of HTLV-1 recognized by class I MHC-restricted cytotoxic T lymphocytes in patients with myelopathy and spastic paraparesis and infected patients with autoimmune disorders.

Human T-cell lymphotropic virus type I (HTLV-1) causes adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The different patterns of clinical diseases are thought to be linked to immunogenetic host factors. A variety of autoimmune diseases, such as Sjögren's syndrome, have been reported in persons infected with HTLV-1, although the precise relationship between these disorders and HTLV-1 infection remains unknown. There is no report on the repertoire of HTLV-1-specific CD8+ T-cells in HAM/TSP patients or carriers with autoimmune diseases, both characterized by an abnormal immune state. In this study, to characterize HTLV-1-specific CD8+ T-cells in asymptomatic HTLV-1 carriers, HAM/TSP patients and carriers with autoimmune diseases, we examined the frequency and diversity of HTLV-1-specific CD8+ T-cells using HTLV-1 tetramers. HTLV-1 Env-specific CD8+ T-cells were significantly more frequent in HAM/TSP and carriers with autoimmune diseases compared with asymptomatic HTLV-1 carriers, while the frequency of HTLV-1 Tax-specific CD8+ T-cells was not significantly different among them. CD8+ cells binding to HTLV-1 Tax tetramers in carriers with autoimmune diseases were significantly reduced compared with HAM/TSP patients. This study demonstrates the importance of CD8+ T-cells recognizing HTLV-1 Env-tetramers in HAM/TSP patients and carriers with autoimmune diseases, thereby suggesting that the diversity, frequency and repertoire of HTLV-1 Env-specific CD8+ T-cell clones may be related to the hyperimmune response in HAM/TSP and carriers with autoimmune diseases, although different immunological mechanisms may mediate the hyperimmunity in these conditions.

Bezafibrate induces plasminogen activator inhibitor-1 gene expression in a CLOCK-dependent circadian manner.

A functional interaction between peroxisome proliferator-activated receptor alpha (PPARalpha) and components of the circadian clock has been suggested, but whether these transcriptional factors interact to regulate the expression of their target genes remains obscure. Here we used a PPARalpha ligand, bezafibrate, to search for PPARalpha-regulated genes that are expressed in a CLOCK-dependent circadian manner. Microarray analyses using hepatic RNA isolated from bezafibrate treated-wild type, Clock mutant (Clk/Clk), and PPARalpha-null mice revealed that 136 genes are transcriptionally regulated by PPARalpha in a CLOCK-dependent manner. Among them, we focused on the plasminogen activator inhibitor-1 (PAI-1) gene, because its expression typically shows circadian variation, and it has transcriptional response elements for both PPAR and CLOCK. The bezafibrate-induced expression of PAI-1 mRNA was attenuated in Clk/Clk mice and in PPARalpha-null mice. The protein levels of PPARalpha were reduced in Clk/Clk hepatocytes. However, the overexpression of PPARalpha could not rescue bezafibrate-induced PAI-1 expression in Clk/Clk hepatocytes, suggesting that impaired bezafibrate-induced PAI-1 expression in Clk/Clk mice is not due to reduced PPARalpha expression. Luciferase reporter and chromatin immunoprecipitation analyses using primary hepatocytes demonstrated that DNA binding of both PPARalpha and CLOCK is essential for bezafibrate-induced PAI-1 gene expression. Pull-down assays in vitro showed that both PPARalpha and its heterodimerized partner retinoic acid receptor-alpha can serve as potential interaction targets of CLOCK. The present findings revealed that molecular interaction between the circadian clock and the lipid metabolism regulator affects the bezafibrate-induced gene expression.

Fasting promotes the expression of SIRT1, an NAD+ -dependent protein deacetylase, via activation of PPARalpha in mice.

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD(+)/NADH ratio in cells and results in activation of SIRT1, an NAD(+)-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARalpha, PPARgamma, and PPARbeta/delta, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARgamma by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARalpha correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARalpha in the livers, where the NAD(+) levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD(+) salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD(+) or NADH showed that the mRNA expression of both SIRT1 and PPARalpha can be enhanced by addition of NAD(+), and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARalpha agonist suggested that PPARalpha is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.

Molecular mechanism for neuro-protective effect of prosaposin against oxidative stress: its regulation of dimeric transcription factor formation.

Prosaposin triggers G-protein-coupled receptor (GPCR)-mediated protein kinase B (Akt)/extracellular signal-regulated kinase (ERK) phosphorylation cascades to exert its neurotrophic and myelinotrophic activity capable of preventing neural cell death and promoting neural proliferation and glial differentiation. In the present study, we investigated the down-stream neurotrophic signaling mechanism of prosaposin by which rat pheochromocytoma (PC-12) cells are protected from cell death induced by oxidative stress. When PC-12 cells were exposed to H2O2, the cells underwent abrupt shrinkage followed by apoptosis. Prosaposin treatment at as low as 1 nM protected PC-12 cells from cell death by the oxidative stress with the activation of an ERK phosphorylation cascade. Simultaneously, prosaposin blocked the oxidative stress induced-Akt phosphorylation that acts on the down-stream of caspase-3 activation. A MEK inhibitor, PD98059, or a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, abolished the survival effect of prosaposin on the oxidative stress-induced cell death. Furthermore, prosaposin blocked the oxidative stress-induced phosphorylations of c-Jun N-terminal kinase (JNK) and p38 stress-activated protein kinase. We further investigated the effect of prosaposin treatment on the phosphorylation of activating protein-1 (AP-1) complex components, c-Jun and activating transcription factor (ATF)-3. Western blot analysis demonstrated that prosaposin treatment at 100 ng/ml decreased the levels of c-Jun and ATF-3 induced by H2O2 stimulation. Our results suggest that prosaposin aids survival of PC-12 cells from oxidative stress not only by reducing the phosphorylation levels of JNK and p38, but also by regulating the c-Jun/AP-1 pathway.

Diastereoselective synthesis of gamma-amino-delta-hydroxy-alpha,alpha-difluorophosphonates: a vehicle for structure-activity relationship studies on SMA-7, a potent sphingomyelinase inhibitor.

A highly diastereoselective synthesis of 2-amino alcohol derivatives bearing a difluoromethylphosphonothioate group at the 3-position was achieved through LiAlH(O-t-Bu)(3)-mediated reduction of the corresponding alpha-amino ketones. The phosphonothioate moiety of the product was readily converted into the corresponding phosphonate by oxidation with m-CPBA, followed by aqueous workup. The developed methods should be useful for SAR studies of SMA-7, a potent inhibitor of SMases.

Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo.

Crocus sativus L. (saffron) has been used as a spice for flavoring and coloring food preparations, and in Chinese traditional medicine as an anodyne or tranquilizer. Our previous study demonstrated that crocin, a carotenoid pigment of saffron, can suppress the serum deprivation-induced death of PC12 cells by increasing glutathione (GSH) synthesis and thus inhibiting neutral sphingomyelinase (nSMase) activity and ceramide formation. The carotenoid pigments of saffron consist of crocetin di-(beta-d-glucosyl)-ester [dicrocin], crocetin-(beta-d-gentiobiosyl)-(beta-d-glucosyl)-ester [tricrocin] and crocetin-di-(beta-d-gentiobiosyl)-ester [crocin]. Saffron also contains picrocrocin, the substance causing saffron's bitter taste. In this study, to confirm whether neuroprotective effects of saffron are caused solely by crocin, we examined the antioxidant and GSH-synthetic activities of these crocins in PC12 cells under serum-free and hypoxic conditions. Measurements of cell viability, peroxidized membrane lipids and caspase-3 activity showed that the rank order of the neuroprotective potency at a concentration of 10 muM was crocin>tricrocin>dicrocin and picrocrocin (the latter two crocins had a little or no potency). In addition, we show that among these saffron's constituents, crocin most effectively promotes mRNA expression of gamma-glutamylcysteinyl synthase (gamma-GCS), which contributes to GSH synthesis as the rate-limiting enzyme, and that the carotenoid can significantly reduce infarcted areas caused by occlusion of the middle cerebral artery (MCA) in mice.

Anti-apoptotic roles of plasminogen activator inhibitor-1 as a neurotrophic factor in the central nervous system.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin gene family, is the primary inhibitor of urokinase-type and tissue-type PAs. PAI-1 plays an important role in the process of peripheral tissue remodeling and fibrinolysis through the regulation of PA activity. This serpin is also produced in brain tissues and may regulate the neural protease sequence in the central nervous system (CNS), as it does in peripheral tissues. In fact, PAI-1 mRNA is up-regulated in mouse brain after stroke. The serpin activity of PAI-1 helps to prevent tissue-type PA-induced neuron death. However, we have previously found that PAI-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. In neuronally differentiated rat pheochromocytoma (PC-12) cells, a deficiency of PAI-1 in vitro caused a significant reduction in Bcl-2 and Bcl-X(L) mRNAs and an increase in Bcl-X(S) and Bax mRNAs. The change in the balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins promoted the apoptotic sequence: caspase-3 activation, cytochrome c release from mitochondria and DNA fragmentation. Our results indicate that PAI-1 has an anti-apoptotic role in neurons. PAI-1 prevented the disintegration of the formed neuronal networks by maintaining or promoting neuroprotective signaling through the MAPK/ERK pathway, suggesting that the neuroprotective effect of PAI-1 is independent of its action as a protease inhibitor. This review discusses the neuroprotective effects of PAI-1 in vitro, together with the relevant data from other laboratories. Special emphasis is placed on its action on PC-12 cells.

Chronic treatment with prednisolone represses the circadian oscillation of clock gene expression in mouse peripheral tissues.

Although altered homeostatic regulation, including disturbance of 24-h rhythms, is often observed in the patients undergoing glucocorticoid therapy, the mechanisms underlying the disturbance remains poorly understood. We report here that chronic treatment with a synthetic glucocorticoid, prednisolone (PSL), can cause alteration of circadian clock function at molecular level. Treatment of cultured hepatic cells (HepG2) with PSL induced expression of Period1 (Per1), and the PSL treatment also attenuated the serum-induced oscillations in the expression of Period2 (Per2), Rev-erbalpha, and Bmal1 mRNA in HepG2 cells. Because the attenuation of clock gene oscillations was blocked by pretreating the cells with a Per1 antisense phosphothioate oligodeoxynucleotide, the extensive expression of Per1 induced by PSL may have resulted in the reduced amplitude of other clock gene oscillations. Continuous administration of PSL into mice constitutively increased the Per1 mRNA levels in liver and skeletal muscle, which seems to attenuate the oscillation in the expressions of Per2, Rev-erbalpha, and Bmal1. However, a single daily administration of PSL at the time of day corresponding to acrophase of endogenous glucocorticoid levels had little effect on the rhythmic expression of clock genes. These results suggest a possible pharmacological action by PSL on the core circadian oscillation mechanism and indicate the possibility that the alteration of clock function induced by PSL can be avoided by optimizing the dosing schedule.

Glucocorticoid regulation of 24-hour oscillation in interferon receptor gene expression in mouse liver.

Although the antiviral effect of interferon (IFN) varies depending on 24-h oscillation in the expression of its specific receptor, the mechanism of oscillation remains to be clarified. Here we report that oscillation in the expression of the IFN receptor gene (IFN-alpha/beta R1) in mouse liver is caused by the endogenous rhythm of glucocorticoid secretion. Brief exposure of mouse hepatic cells (Hepa 1-6) to corticosterone (CORT) resulted in a significant decrease in mRNA levels of IFN-alpha/beta R1. The CORT-induced decrease in IFN-alpha/beta R1 mRNA levels was reversed by pretreating the cells with RU486, a glucocorticoid receptor antagonist. The mRNA levels of IFN-alpha/beta R1 gene in the liver of adrenalectomized mice were consistently increased throughout the day. However, a single administration of CORT to adrenalectomized mice significantly decreased the mRNA levels of IFN-alpha/beta R1 in the liver. Furthermore, the rhythmic phase of IFN-alpha/beta R1 expression was modulated after the alteration of rhythmicity in glucocorticoid secretion, which was induced by restricted daily feeding. As a consequence, under manipulation of the feeding schedule, 2'-5' oligoadenylate synthase activities, as an index of antiviral effect, in plasma and liver at 24 h after IFN-alpha injection also varied depending on the alteration of glucocorticoid secretion rhythm. These results suggest that the endogenous rhythm of glucocorticoid secretion is involved in the circadian regulation of IFN-alpha/beta R1 expression in mouse liver. Our findings also support the notion that monitoring the 24-h variation in IFN receptor function is useful for selecting the most appropriate time of day to administer IFN.

Circadian regulation of mouse topoisomerase I gene expression by glucocorticoid hormones.

Because glucocorticoid hormones modulate various biological processes, the endogenous rhythm of their secretion is thought to be an important factor affecting the efficacy and/or toxicity of many drugs. Topoisomerase I (Topo I) is a nuclear target of the anticancer drug camptothecin (CPT). In this study, we demonstrate that Topo I expression in tumor-bearing mice and the efficacy of CPT on the tumor are affected by the 24-h variation in circulating glucocorticoid levels. A single administration of corticosterone (CORT) to the tumor-bearing mice resulted in a significant increase in Topo I mRNA levels not only in the tumor masses but also in other healthy tissues such as liver and skeletal muscle. The CORT-induced increase in Topo I mRNA was suppressed by pretreating the mice with RU486, a glucocorticoid receptor antagonist. Significant 24-h oscillations in the Topo I mRNA levels were observed in the tumor and healthy liver without exogenous CORT, and were eliminated by adrenalectomy of the mice. This result suggests that endogenous glucocorticoid hormones are involved in the circadian regulation of Topo I gene expression. Furthermore, the anti-tumor efficacy of the Topo I inhibitor CPT-11 on the tumor-bearing mice was enhanced by administering the drug at the time when the Topo I activity was increased. Our present results demonstrate that glucocorticoid is involved in the 24-h oscillation mechanism of Topo I gene expression and suggest that monitoring the circadian rhythm in Topo I activity is useful for choosing the most appropriate time of day to administer of Topo I inhibitors.

A molecular mechanism regulating circadian expression of vascular endothelial growth factor in tumor cells.

Because angiogenesis is essential for tumor growth and metastasis, inhibition of angiogenesis has emerged as a new therapy to treat cancers. Hypoxia-induced expression of vascular endothelial growth factor (VEGF) plays a central role in tumor-induced angiogenesis. In this study, we found that expression of VEGF in hypoxic tumor cells was affected by the circadian organization of molecular clockwork. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop in which CLOCK and BMAL1 are positive regulators, and Period and Cryptochrome genes act as negative ones. The levels of VEGF mRNA in tumor cells implanted in mice rose substantially in response to hypoxia, but the levels fluctuated rhythmically in a circadian fashion. Luciferase reporter gene analysis revealed that Period2 and Cryptochrome1, whose expression in the implanted tumor cells showed a circadian oscillation, inhibited the hypoxia-induced VEGF promoter activity. These results suggest that the negative limbs of the molecular loop periodically inhibit the hypoxic induction of VEGF transcription, resulting in the circadian fluctuation of its mRNA expression. We also showed that the antitumor efficacy of antiangiogenic agents could be enhanced by administering the drugs at the time when VEGF production increased. These findings support the notion that monitoring of the circadian rhythm in VEGF production is useful for choosing the most appropriate time of day for administration of antiangiogenic agents.

24-hour oscillation of mouse methionine aminopeptidase2, a regulator of tumor progression, is regulated by clock gene proteins.

Methionine aminopeptidase2 (MetAP2) plays an important role in the growth of endothelial cells during the tumor angiogenesis stage. Recently, we have clarified that mouse methionine aminopeptidases (mMetAPs) show a 24-hour rhythm in implanted tumor masses. In the present study, we investigated the mechanism underlying the 24-hour rhythm of mMetAP2 activity in tumor-bearing mice under a light-dark (lights on from 7 a.m. to 7 p.m.) cycle. The 5' flanking region of mMetAP2 included eight E-boxes. The transcription of the mMetAP2 promoter was enhanced by the mCLOCK:mBMAL1 heterodimer, and its activation was inhibited by mPER2 or mCRY1. Deletion and mutation of the E-boxes in the region indicated that the E-box nearest to the initiation start site played an important role in the transcriptional regulation by clock genes. In sarcoma180-bearing mice, the pattern of binding of mCLOCK and mBMAL1 to the E-box and transcription of the mMetAP2 promoter showed a 24-hour rhythm with higher levels from the mid-light to early dark phase. The pattern of mMetAP2 transcription was closely associated with that of mMetAP2 mRNA expression in three types of tumor-bearing mice. mMetAP2 protein expression varied with higher levels from the late-dark to early light phase. The rhythmicity of the protein expression was synchronous with that of the activity of mMetAPs but out of phase with that of the mMetAP2 mRNA expression. These results suggest that the 24-hour rhythm of mMetAP2 activity is regulated by the transcription of clock genes within the clock feedback loops.

An attempt to promote neo-vascularization by employing a newly synthesized inhibitor of protein tyrosine phosphatase.

Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor-2 (VEGFR-2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR-2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of alpha,alpha-difluoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their effects on VEGF-induced angiogenesis. DFPMPA-3, the most effective inhibitor of human PTP-1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more effectively than any other DFPMPAs. The inhibitor promoted the VEGF-induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR-2. Its effectiveness was proven through neo-vascularization in mice. The present findings suggest that targeting PTP to promote therapeutic neo-vascularization may be a potential strategy.

Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities.

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has anticoagulant and antithrombotic activities. Unlike heparin, it shows an inhibitory action on the progression and metastasis of malignant tumors, although the precise mechanisms have not been elucidated. We have demonstrated previously that fucoidan can inhibit tube formation following migration of human umbilical vein endothelial cells (HUVEC) and that its chemical oversulfation enhances the inhibitory potency. In this study, we tested the hypothesis that fucoidan may suppress tumor growth by inhibiting tumor-induced angiogenesis. Both natural and oversulfated fucoidans (NF and OSF) significantly suppressed the mitogenic and chemotactic actions of vascular endothelial growth factor 165 (VEGF(165)) on HUVEC by preventing the binding of VEGF(165) to its cell surface receptor. The suppressive effect of OSF was more potent than that of NF, suggesting an important role for the numbers of sulfate groups in the fucoidan molecule. Consistent with its inhibitory actions on VEGF(165), OSF clearly suppressed the neovascularization induced by Sarcoma 180 cells that had been implanted in mice. The inhibitory action of fucoidan was also observed in the growth of Lewis lung carcinoma and B16 melanoma in mice. These results indicate that the antitumor action of fucoidan is due, at least in part, to its anti-angiogenic potency and that increasing the number of sulfate groups in the fucoidan molecule contributes to the effectiveness of its anti-angiogenic and antitumor activities.

Crocin prevents the death of PC-12 cells through sphingomyelinase-ceramide signaling by increasing glutathione synthesis.

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) that has been used in traditional Chinese medicine. In a previous study, we demonstrated that crocin inhibits apoptosis in PC-12 cells by affecting the function of tumor necrosis factor-alpha. In this study, we found that depriving cultured PC-12 cells of serum/glucose causes a rapid increase in cellular ceramide levels, followed by an increase in the phosphorylation of c-jun kinase (JNK). The accumulation of ceramide was found to depend on the activation of magnesium-dependent neutral sphingomyelinase (N-SMase), but not on de novo synthesis. The serum/glucose-deprived PC-12 cells also decreased the cellular levels of glutathione (GSH), which is the potent inhibitor of N-SMase. Treating the PC-12 cells with crocin prevented N-SMase activation, ceramide production, and JNK phosphorylation. We also found that the chemical can enhance the activities of GSH reductase and gamma-glutamylcysteinyl synthase (gamma-GCS), contributing to a stable GSH supply that blocks the activation of N-SMase. Thus our data suggest that crocin combats the serum/glucose deprivation-induced ceramide formation in PC-12 cells by increasing GSH levels and prevents the activation of JNK pathway, which is reported to have a role of the signaling cascade downstream ceramide for neuronal cell death.

Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress.

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.

Plasminogen activator inhibitor-1 aids survival of neurites on neurons derived from pheochromocytoma (PC-12) cells.

Plasminogen activator inhibitor-1 is a serpin that regulates the activities of plasminogen activators. However, its physiological roles in the CNS are incompletely understood. We have found that plasminogen activator inhibitor-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. PC-12 cells treated with nerve growth factor differentiated into neurons and formed a network of neurites. In a serum-free culture medium, these neurites disappeared within 24 h. The addition of plasminogen activator inhibitor-1 prevented the disintegration of the neuronal networks, while the addition of the serpin inhibitors aprotinin and antipain did not. The plasminogen activator inhibitor-1 maintained or promoted the phosphorylated state of extracellular signal-regulated kinase (ERK), but not of protein kinase B (Akt). These results are the first evidence that plasminogen activator inhibitor-1 in the CNS acts to maintain the morphology of neurites via activation of the ERK-related pathway in the neurons.

Crocin prevents the death of rat pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of alpha-tocopherol.

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) used in traditional Chinese medicine. We report here the effects of crocin on neuronally differentiated pheochromocytoma (PC-12) cells deprived of serum/glucose. Depriving the PC-12 cells of serum/glucose caused peroxidation of their cell membrane lipids and decreased intercellular superoxide dismutase (SOD) activity. Treating the PC-12 cells with 10 microM crocin inhibited the formation of peroxidized lipids, partly restored the SOD activity, and maintained the neuron's morphology. These antioxidant effects of crocin were more effective than those of alpha-tocopherol at the same concentration. Crocin also suppressed the activation of caspase-8 caused by serum/glucose deprivation. These results together with our previous data suggest that crocin is a unique and potent antioxidant that combats oxidative stress in neurons.

Covalent binding of nitroso-sulfonamides to glutathione S-transferase in guinea pigs with delayed type hypersensitivity.

Drug induced allergies are believed to be induced by conjugates consisting of biological macromolecules and active metabolites. The present study investigated whether guinea pig glutathione S-transferase (gpGST), a protein that binds with sulfanilamide (SA) and sulfamethoxazole (SMX), could be detected in the liver cytosol fraction of guinea pigs that intraperitoneally received SA or SMX, and whether gpGST is a carrier protein. We synthesized three nitroso compounds, i.e., 4-nitroso-sulfanilamide (SA-NO), 4-nitrososulfamethoxazole (SMX-NO) and fluorescent-labeled nitroso compound (DNSBA-NO), and examined binding quantities of nitroso compounds to gpGST purified from untreated female guinea pigs. Furthermore, the concentrations of IgG in serum antibody for nitroso compounds were estimated using ELISA. When guinea pigs were sensitized using the three nitroso compounds, the dose dependent skin reactions were confirmed with each compound. In addition, sensitized guinea pigs using each nitroso compound showed positive skin reactions at an elicitation test performed using gpGST alone. The results confirmed synthesis of antibody against gpGST due to hapten sensitization. Therefore, when a nitroso compound binds with gpGST in the body of guinea pigs, nitroso-gpGST acts as a neoantigen, which induces synthesis of autoantibody. Thus, gpGST appears to be one of the carrier proteins that induce sulfa drug-induced allergies. Immunization of guinea pigs with active metabolite of drugs may give information for predicting the occurrence of delayed type hypersensitivity in human.