The small molecule STF-62247 induces apoptotic and autophagic cell death in leukemic cells.

Adult T cell leukemia/lymphoma (ATL) is an aggressive malignant T cell disease caused by human T cell leukemia virus-I (HTLV-1). Treatment outcomes for aggressive subtypes of ATL remain poor, with little improvement in overall survival since HTLV-1 was discovered. Therefore, new therapeutic strategies for ATL are required. STF-62247 induces autophagy and selectively kills renal cell carcinoma without apoptotic cell death. Here, we demonstrate that STF-62247 reduced cell viability and resulted in autophagosome accumulation and autophagy in leukemic cell lines (S1T, MT-2, and Jurkat). Interestingly, STF-62247 induced apoptosis in HTLV-1-infected cell lines (S1T and MT-2), as indicated by DNA fragmentation and caspase activation, but not in non-HTLV-1-infected Jurkat cells; a caspase inhibitor did not prevent this caspase-associated cell death. STF-62247 also increased nuclear endonuclease G levels. Furthermore, STF-62247 reduced cell viability and increased the number of apoptotic cells in peripheral blood mononuclear cells collected from patients with acute ATL, which has a poor prognosis. Therefore, STF-62247 may have novel therapeutic potential for ATL. This is the first evidence to demonstrate the cell growth-inhibitory effect of an autophagy inducer by caspase-dependent apoptosis and caspase-independent cell death via autophagy and endonuclease G in leukemic cells.

Neuroprotective Activities of Saffron and Crocin.

We first considered that saffron is really safety food because it has a long-use history. The neuroprotective activities of saffron and its major constituent, crocin, are separately discussed in vitro and in vivo. We reviewed the inhibitory activities of crocin against PC-12 cell apoptosis. The oxidative stress decreased the cellular levels of glutathione (GSH) which is an inhibitor of neutral sphingomyelinase (N-SMase). Therefore, the level of GSH was assayed by the addition of crocin resulted in the activation of glutathione reductase (GR). It became evident that crocin treatment prevents the N-SMase activation resulting in the decrease of ceramide release. From these evidences we summarized the role of crocin for neuronal cell death. We used the ethanol-blocking assay system for learning and memory activities. The effect of saffron and crocin on improving ethanol-induced impairment of learning behaviors of mice in passive avoidance tasks has been clear. Further, we did make clear that saffron and crocin prevent the inhibitory effect of ethanol on long-term potentiation (LTP) in the dentate gyrus. Finally we found that 100 mg/kg of crocin gave non-rapid eye movement sleep (non-REM sleep) although mice were started to be active during night time.

Angiotensin II type 1 receptor blocker telmisartan induces apoptosis and autophagy in adult T-cell leukemia cells.

Adult T-cell leukemia/lymphoma (ATL), an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1), requires new treatments. Drug repositioning, reuse of a drug previously approved for the treatment of another condition to treat ATL, offers the possibility of reduced time and risk. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate peroxisome proliferator-activated receptor-γ, which plays various roles in lipid metabolism, cellular differentiation, and apoptosis. Here, telmisartan reduced cell viability and enhanced apoptotic cells via caspase activation in ex vivo peripheral blood monocytes from asymptomatic HTLV-1 carriers (ACs) or via caspase-independent cell death in acute-type ATL, which has a poor prognosis. Telmisartan also induced significant growth inhibition and apoptosis in leukemia cell lines via caspase activation, whereas other angiotensin II receptor blockers did not induce cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation and autophagy. Thus, telmisartan simultaneously caused caspase activation and autophagy. A hypertension medication with antiproliferation effects on primary and leukemia cells is intriguing. Patients with an early diagnosis of ATL are generally monitored until the disease progresses; thus, suppression of progression from AC and indolent ATL to acute ATL is important. Our results suggest that telmisartan is highly effective against primary cells and leukemia cell lines in caspase-dependent and -independent manners, and its clinical use may suppress acute transformation and improve prognosis of patients with this mortal disease. This is the first report demonstrating a cell growth-inhibitory effect of telmisartan in fresh peripheral blood mononuclear cells from leukemia patients.

Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells.

Adult T-cell leukaemia/lymphoma (ATL) is an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukaemia virus (HTLV)-1. The identification of new molecular targets for ATL prevention and treatment is desired. SIRT1, a nicotinamide adenine dinucleotide(+) -dependent histone/protein deacetylase, plays crucial roles in various physiological processes, including aging and apoptosis. We previously reported that ATL patients had significantly higher SIRT1 protein levels than healthy controls. Here, we demonstrate that two novel small-molecule SIRT1 inhibitors, NCO-01/04, reduced cell viability and enhanced apoptotic cells in peripheral blood monocyte cells of patients with acute ATL, which has a poor prognosis. NCO-01/04 also reduced the cell viability with DNA fragmentation, Annexin V-positive cells, and caspase activation. However, a caspase inhibitor did not inhibit this caspase-dependent cell death. NCO-01/04 enhanced the endonuclease G level in the nucleus with loss of the mitochondrial transmembrane potential, which can promote caspase-independent death. Interestingly, NCO-01/04 increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Thus, NCO-01/04 simultaneously caused caspase activation and autophagy. These results suggest that NCO-01/04 is highly effective against ATL cells in caspase-dependent or -independent manners with autophagy, and that its clinical application might improve the prognosis of patients with this fatal disease.

Effect of adenosine system in the action of oseltamivir on behavior in mice.

Abnormal behaviors and death associated with the use of oseltamivir (Tamiflu(®)) have emerged as a major issue in influenza patients. We have previously reported that the mechanisms underlying the effects of caffeine, a non-selective adenosine A1/A2 receptor antagonist, combined with oseltamivir. Oseltamivir is rapidly hydrolyzed to its active form (oseltamivir carboxylate, OCB). In this study, we investigated the effects of an adenosine system and OCB on the action of oseltamivir on mice behavior. Oseltamivir for 1 day (150 mg/kg, intraperitoneally (i.p.)) alone did not affect ambulation at 2 h post-injection. However, caffeine (10 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, caffeine (30 mg/kg, i.p.) in combination with oseltamivir for 3 days increased ambulation, but caffeine (10 mg/kg, i.p.) in combination with oseltamivir for 3 days did not increase. These enhancements were inhibited by an adenosine A2 receptor agonist, CGS21680 (0.2 mg/kg, subcutaneously (s.c.)). Furthermore, an adenosine A2 receptor antagonist, SCH58261 (1 and 3 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, SCH58261 (3 mg/kg, i.p.) in combination with oseltamivir for 3 days increased ambulation, but SCH58261 (1 mg/kg, i.p.) in combination with oseltamivir for 3 days did not. Conversely, in phenobarbital (PB)-treated mice, caffeine (3 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, OCB for 1 day (0.3 µg/mouse intracerebroventricular (i.c.v.)) alone increased ambulation. These findings suggest that the actions of oseltamivir may involve the adenosine systems and its metabolism. Our findings suggest an interaction between the central blockade of adenosine A2 receptors by caffeine and OCB-induced behavioral changes.

Anticancer agents targeted to sirtuins.

Sirtuins are nicotinamide adenine dinucleotide+-dependent deacetylases of which there are seven isoforms (SIRT1-7). Sirtuin activity is linked to gene expression, lifespan extension, neurodegeneration, and age-related disorders. Numerous studies have suggested that sirtuins could be of great significance with regard to both antiaging and tumorigenesis, depending on its targets in specific signaling pathways or in specific cancers. Recent studies have identified small chemical compounds that modulate sirtuins, and these modulators have enabled a greater understanding of the biological function and molecular mechanisms of sirtuins. This review highlights the possibility of sirtuins, especially SIRT1 and SIRT2, for cancer therapy targets, and focuses on the therapeutic potential of sirtuin modulators both in cancer prevention and treatment.

The evaluation method for antiplatelet effect of acetylsalicylic acid.

Reduced platelet aggregation by acetylsalicylic acid administration has been associated with adverse outcomes in patients with thrombotic diseases, thus it is important to determine aspirin resistance in those cases. The antiplatelet effect of acetylsalicylic acid is rarely measured, but it has many problems. The aim of this study was to find the evaluation method for antiplatelet effect after administration of acetylsalicylic acid. We developed a particle counting method based upon laser light scattering, and utilized the platelet aggregation agonists, collagen, at 0.25, 0.5 and 1.0 µg/mL, and adenosine diphosphate (ADP), at 0.5, 1.0 and 2.0 µM, to determine their effective concentrations. Seventeen healthy volunteers were administered acetylsalicylic acid at 162 mg/day, with platelet aggregation determined before and 20 min after administration. In all subjects, the rate of platelet aggregation induced by 1.0 µg/mL of collagen before taking acetylsalicylic acid was the highest value obtained, while 20 min after acetylsalicylic acid administration, aggregation induced by collagen at 1.0 µg/mL was significantly decreased as compared to before administration. As for the other concentrations of collagen and all those of ADP tested, platelet aggregation was either not significantly induced before taking acetylsalicylic acid or the rate of aggregation was not significantly decreased after taking acetylsalicylic acid. Our results indicate that collagen at 1.0 µg/mL is appropriate as a platelet aggregation agonist for evaluating the antiplatelet effect of acetylsalicylic acid. Thus, it is useful that the measurement is performed only once.

Liposomes and nanotechnology in drug development: focus on oncotargets.

Nanotechnology is the development of an engineered device at the atomic, molecular, and macromolecular level in the nanometer range. Advances in nanotechnology have proven beneficial in therapeutic fields such as drug-delivery and gene/protein delivery. Antigen delivery systems are important for inducing and modifying immune responses. In cellular immunity, cytotoxic T lymphocytes (CTLs) are important in the host defense against tumors. Key to the development of CTL-inducible vaccines is the ability to deliver antigens to antigen-presenting cells efficiently and to induce the subsequent activation of T cell-mediated immunity without adjuvants, as they can induce excessive inflammation leading to systemic febrile disease. Since expression and cloning methods for tumor-associated antigens have been reported, cancer vaccines that induce effective cell immunity may be promising therapeutic candidates, but Th2 cells are undesirable for use in cancer immunotherapy. Peptide vaccines have immunological and economic advantages as cancer vaccines because CTL epitope peptides from tumor-associated antigens have high antigen-specificity. However, cancer vaccines have had limited effectiveness in clinical responses due to the ability of cancer cells to "escape" from cancer immunity and a low efficiency of antigen-specific CTL induction due to immunogenic-free synthetic peptides. In contrast, carbohydrate-decorated particles such as carbohydrate-coated liposomes with encapsulated antigens might be more suitable as antigen delivery vehicles to antigen-presenting cells. Oligomannose-coated liposomes (OML) can eliminate established tumors in mouse cancer models. In addition, OMLs with an encased antigen can induce antigen-specific CTLs from peripheral blood mononuclear cells obtained from patients. Feasibility studies of OML-based vaccines have revealed their potential for clinical use as vaccines for diseases where CTLs act as effector cells. Furthermore, use of the hepatitis B core particle, in which tumor-antigen epitopes are set, has consistently been shown to induce strong CTL responses without the use of an adjuvant. Thus, nanoparticles may provide a new prophylactic strategy for infectious disease and therapeutic approaches for cancer via the induction of T-cell immunity.

Vasohibin induces prolyl hydroxylase-mediated degradation of hypoxia-inducible factor-1α in human umbilical vein endothelial cells.

Vasohibin is thought to be an important negative feedback regulator of angiogenesis that is selectively induced in endothelial cells by VEGF. Here, we assessed the role of vasohibin on HIF-1α expression under oxidative stress induced by hydrogen peroxide (H2O2) in HUVEC. VEGF induced significant cell growth that was associated with an increase in vasohibin expression. Following H2O2-pretreatment, VEGF further increased cell growth but this was contrastingly associated with a decrease in vasohibin expression when compared with VEGF alone. Interestingly, vasohibin inhibited cell proliferation through degradation of HIF-1α expression during H2O2-pretreatment. Furthermore, vasohibin elevated the expression of prolyl hydroxylase (PHD). These results suggest that vasohibin plays crucial roles as a negative feedback regulator of angiogenesis through HIF-1α degradation via PHD.

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.

Programmed death-1 (PD-1)/PD-1 ligand pathway-mediated immune responses against human T-lymphotropic virus type 1 (HTLV-1) in HTLV-1-associated myelopathy/tropical spastic paraparesis and carriers with autoimmune disorders.

Human T-lymphotropic virus-1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia-lymphoma in individuals with dysfunctional immune responses. In this study, to characterize the HTLV-1-specific cytotoxic T lymphocyte (CTL) populations in asymptomatic HTLV-1 carriers (ACs), HAM/TSP patients, and carriers with autoimmune disorders (CAIDs), we examined the role of programmed death-1 and its ligand (PD-1/PD-L1) in HTLV-1-specific CTL functions using an HTLV-1 Tax/HLA-A*0201 tetramer and an HTLV-1 Tax/HLA-A*2402 tetramer. Interestingly, the percentage of HTLV-1 Tax301-309/HLA-A*2402 tetramer(+)CD8(+) cells expressing PD-1 in ACs was significantly higher than the percentage of HTLV-1 Tax11-19/HLA-A*0201 tetramer(+)CD8(+) cells expressing PD-1. PD-1 expression was significantly downregulated on HTLV-1-specific CTLs in HAM/TSP compared with ACs. PD-L1 expression was observed in a small proportion of unstimulated lymphocytes from ACs and was greater in ACs than in HAM/TSP and CAIDs after short-term culture. Furthermore, CTL degranulation was impaired in HAM/TSP, whereas anti-PD-L1 blockade significantly increased CTL function in ACs. Downregulation of PD-1 on HTLV-1-specific CTLs and loss of PD-L1 expression in HAM/TSP and CAIDs, along with impaired function of HTLV-1-specific CTLs in HAM/TSP, may underlie the apparently dysfunctional immune response against HTLV-1.

Oligomannose-coated liposomes efficiently induce human T-cell leukemia virus-1-specific cytotoxic T lymphocytes without adjuvant.

Human T-cell leukemia virus-1 (HTLV-1) causes adult T-cell leukemia/lymphoma, which is an aggressive peripheral T-cell neoplasm. Insufficient T-cell response to HTLV-1 is a potential risk factor in adult T-cell leukemia/lymphoma. Efficient induction of antigen-specific cytotoxic T lymphocytes is important for immunological suppression of virus-infected cell proliferation and oncogenesis, but efficient induction of antigen-specific cytotoxic T lymphocytes has evaded strategies utilizing poorly immunogenic free synthetic peptides. Here, we examined the efficient induction of an HTLV-1-specific CD8+ T-cell response by oligomannose-coated liposomes (OMLs) encapsulating the human leukocyte antigen (HLA)-A*0201-restricted HTLV-1 Tax-epitope (OML/Tax). Immunization of HLA-A*0201 transgenic mice with OML/Tax induced an HTLV-1-specific gamma-interferon reaction, whereas immunization with epitope peptide alone induced no reaction. Upon exposure of dendritic cells to OML/Tax, the levels of CD86, major histocompatibility complex class I, HLA-A02 and major histocompatibility complex class II expression were increased. In addition, our results showed that HTLV-1-specific CD8+ T cells can be efficiently induced by OML/Tax from HTLV-1 carriers compared with epitope peptide alone, and these HTLV-1-specific CD8+ T cells were able to lyse cells presenting the peptide. These results suggest that OML/Tax is capable of inducing antigen-specific cellular immune responses without adjuvants and may be useful as an effective vaccine carrier for prophylaxis in tumors and infectious diseases by substituting the epitope peptide.

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.

The stimulatory effects of caffeine with oseltamivir (Tamiflu) on light-dark behavior and open-field behavior in mice.

Abnormal behaviors and death associated with the use of oseltamivir (Tamiflu) have emerged as a major issue in influenza patients taking the drug. Here, we investigated the mechanisms underlying the effects of oseltamivir on the behavior of mice using light-dark and open-field preference tests. Oseltamivir (75 and 150 mg/kg, intraperitoneally (i.p.)) alone affected neither time spent in the open area in the light-dark preference test nor ambulation in the open-field test at 2h post-injection. However, a non-selective adenosine A(1)/A(2) receptor antagonist, caffeine (10mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased time spent in the open area in the light-dark preference test. This enhancement was not inhibited by a benzodiazepine receptor antagonist, flumazenil (10-20mg/kg, subcutaneously (s.c.)). Enhancement of ambulation in the open-field test was also observed when caffeine (10mg/kg, i.p.) was combined with oseltamivir (150 mg/kg, i.p.). This enhancement was inhibited by a dopamine D(2) receptor antagonist, haloperidol (0.1mg/kg, s.c.). Furthermore, an adenosine A(2) receptor antagonist, SCH58261 (3mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased ambulation in the open-field test, while an adenosine A(1) receptor antagonist, DPCPX (1-3mg/kg, i.p.) did not. These findings suggest that the actions of oseltamivir may involve the dopamine and adenosine systems. Our findings suggest that due to the interaction between central blockade of adenosine A(2) receptors by caffeine, and oseltamivir-induced behavioral changes, patients being treated with oseltamivir should be closely monitored.

Efficient induction of human T-cell leukemia virus-1-specific CTL by chimeric particle without adjuvant as a prophylactic for adult T-cell leukemia.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with the human T-cell leukemia virus-1 (HTLV-1). HTLV-1-specific cytotoxic T lymphocytes (CTLs) play an important role in suppressing proliferation of HTLV-1-infected or transformed T-cells in vitro. Efficient induction of antigen-specific CTLs is important for immunologic suppression of oncogenesis, but has evaded strategies utilizing poorly immunogenic free synthetic peptides. In the present study, we examined the efficient induction of HTLV-1-specific CD8+ T-cell response by an HTLV-1/hepatitis B virus core (HBc) chimeric particle incorporating the HLA-A*0201-restricted HTLV-1 Tax-epitope. The immunization of HLA-A*0201-transgenic mice with the chimeric particle induced antigen-specific gamma-interferon reaction, whereas immunization with epitope peptide only induced no reaction as assessed by enzyme-linked immunospot assay. Immunization with the chimeric particle also induced HTLV-1-specific CD8+ T-cells in spleen and inguinal lymph nodes. Furthermore, upon exposure of dendritic cells from HLA-A*0201-transgenic mice to the chimeric particle, the expression of CD86, HLA-A02, TLR4 and MHC class II was increased. Additionally, our results show that HTLV-1-specific CD8+ T-cells can be induced by peptide with HTLV-1/HBc particle from ATL patient, but not by peptide only and these HTLV-1-specific CD8+ T-cells were able to lyse cells presenting the peptide. These results suggest that HTLV-1/HBc chimeric particle is capable of inducing strong cellular immune responses without adjuvants via effective maturation of dendritic cells and is potentially useful as an effective carrier for therapeutic vaccines in tumors, or in infectious diseases by substituting the epitope peptide.

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.

[Approach to CAPD services by pharmacists].

In Tokai University Hachioji Hospital, a delivery system of peritoneal dialysis fluid has been built and pharmacists have been coordinating the delivery services. This time, we researched and verified the operation status for two years from April 2007 to March 2009. It was considered that a coordination of operation services for peritoneal dialysis fluid by pharmacists has been able to contribute to "medical safety" and "medical cost reduction". Also for a medical staff, changes in prescription and amount of stock have been able to reduce because the delivery system operation of peritoneal dialysis fluid has penetrated. However, as patients' understanding for the delivery system of peritoneal dialysis fluid was not enough, it was considered as the cause of increase in take-home drugs/days. As for issues in the future, we hope to study methods to enhance patient education at the time of hospital admission and confirm patients' understanding at the time of hospital visits.

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.

Modulation of circadian rhythm of DNA synthesis in tumor cells by inhibiting platelet-derived growth factor signaling.

Circadian synchronization of cell proliferation is observed not only in normal healthy tissues but also in malignant solid tumors. However, the proliferation rhythm of tumor cells is often different from that of normal cells. We reported here that the peculiar rhythm of tumor cell proliferation was modulated by inhibition of platelet-derived growth factor (PDGF) signaling. DNA synthesis in tumor cells implanted in mice showed a 24-h oscillation apparently differing from that of normal bone marrow cells. Continuous administration of AG1295 (10 microg/h, s.c.), a PDGF receptor tyrosine kinase inhibitor, substantially suppressed DNA synthesis in the implanted tumor cells but not in the healthy bone marrow cells. During the administration of this drug, the rhythm of DNA synthesis in the tumor cells was synchronized with that in bone marrow cells. The present results suggest that the circadian rhythm of DNA synthesis in tumor cells is modulated by PDGF receptor signaling, which is activated following tumor progression. Because the rhythmic patterns of clock gene expression in tumor cells did not differ significantly from those in other healthy tissues, the enhanced signal transduction of PDGF receptor may cause an alteration in the rhythmicity of tumor cell proliferation without changing in the intracellular molecular clockwork.

[The role of pharmacist on coordinating CAPD operation].

Tokai University Hachioji Hospital has built a distribution system which intended to minimize a drug inventory at the hospital, and to reduce the number of drugs when the patient is discharged or when the patient is an outpatient to take-out the drugs in order to facilitate smooth CAPD operation. As a result, the number of take-out drugs for discharged patients or outpatients has been reduced within two days. In addition to that, it has been considered to minimize both the strain to the patient who has to take drugs back to home as well as a level of the drug inventory. The cost of peritoneal dialysate inventory amount corresponds to the amount of one patient's 3-day inventory. Therefore, the amount of money can be suppressed to the minimum. Since the distribution system has not caused any major troubles, we basically assumed the system operation to be successful. Although CAPD operation is performed by clinical nurses in most of the cases, we believe that CAPD operation can be smoothly performed by participation of pharmacist. Further more, it is possible to contribute toward a health cost reduction, and the like.