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Therapy with α-radiation has issues associated with internal exposure; its clinical use has been avoided. However, phase III clinical tests of the α-emitting nuclide 223Ra on patients with cancer have been conducted, and results were reported in 2011 to 2012. Since then, research has being carried out on targeted internal therapy by introducing α-emitting nuclides directly into the cancers. For many decades, nontargeted radon therapy has been carried out and is controversial because its mechanism of action is stimulation. The low-level radiation sends powerful signals to upregulate many biological protection systems, which protect against the effects of radiogenic and nonradiogenic toxins. These vital systems prevent, repair, and remove DNA and other biomolecular damage being produced endogenously at a very high rate by the very abundant reactive oxygen species associated with aerobic metabolism. Stimulation of protection systems results in beneficial effects, including a lower risk of cancer. This article reports the results of treatments on 4 patients with cancer and reviews the clinical use of α-radiation from 223Ra and radon. It discusses the prospect of using the novel 225Ac-prostate-specific membrane antigen ligand-617 ligand as a therapeutic agent for prostate cancer. It presents a new treatment system that we developed, α-Radiorespir o-Rn, which seems to be extremely effective in treating cancer.

The transcription factor nuclear erythroid-derived 2-related factor 2 (Nrf2) regulates expression of genes encoding antioxidant proteins involved in cellular redox homeostasis, while gamma-ray irradiation is known to induce reactive oxygen species in vivo. Although activation of Nrf2 by various stresses has been studied, it has not yet been determined whether ionizing irradiation induces activation of Nrf2. Therefore, we investigated activation of Nrf2 in response to gamma-irradiation in mouse macrophage RAW264.7 cells. Irradiation of cells with gamma-rays induced an increase of Nrf2 expression. Even 0.1 Gy of gamma-irradiation induced a translocation of Nrf2 from cytoplasm to the nucleus, indicating the activation of Nrf2 by low-dose irradiation. Expression of heme oxygenase-1, which is regulated by Nrf2, was also increased at 24 h after irradiation with more than 0.1 Gy of gamma-rays. Furthermore, the activation of Nrf2 was suppressed by U0126, which is an inhibitor of the extracellular signal regulated protein kinase 1/2 (ERK1/2) pathway, suggesting involvement of ERK1/2-dependent pathway in the irradiation-induced activation of Nrf2. Our results indicate that low-dose gamma-irradiation induces activation of Nrf2 through ERK1/2-dependent pathways.

癌や老化などのさまざまな疾患では，過剰な活性酸素(O2-・)の発生が深く関係している．生体内で過剰に発生した O2-・ を消去するため，生体内投与を視野に入れ，かつ，長期血中滞留性を有する新規抗酸化剤としてカチオン性マンガンポルフィリン錯体導入ニオソームを調製した．抗酸化作用を有するマンガン 5,10,15,20-テトラキス(N-2-メチルピリジル)ポルフィリン(MnT2MePyP)およびマンガン 5,10,15,20-テトラキス(S,S-4-ジメチルスルフォニオフェニル)ポルフィリン(MnT4Me2SuP)をノニオン性界面活性剤主成分(プルロニック(Pluronic)F-88/コレステロール系)のニオソームに導入した(マンガンポルフィリン/ニオソーム)．調製したマンガンポルフィリン/ニオソームの構造は，動的光散乱および蛍光偏光解消法により確認した．血中投与した際の安定性は棚置き安定性試験および擬似血中滞留試験により評価した．また，抗酸化剤としての性能をストップトフロー法および動物試験により評価した．ストップトフロー法の結果より，マンガンポルフィリン/ニオソームはマンガンポルフィリン単独系とほぼ同等の高い抗酸化活性を示した．マウスを用いた動物実験の結果より，マンガンポルフィリン/ニオソームはマンガンポルフィリン錯体単独系よりも長期血中滞留性を有し，有意な抗酸化活性の向上が見られた．マンガンポルフィリン/ニオソームは抗酸化作用の DDS への応用が期待できる．

Background Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma. Methods Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X 7 receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X 7 receptor. Results Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X 7 receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X 7 receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X 7 receptor channel plays a significant role in mediating the ATP release. General Significance We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.

The effect of sunlight on the development of allergic diseases is not well understood. In this study, we show that increased production of the proinflammatory mediators interleukin (IL)-10, tumor necrosis factor (TNF)-α ,a nd nitric oxide (NO) induced by ultraviolet B (UVB) is mediated via the mitogen-activated protein kinase (MAPK) signaling pathway in human keratinocyte (HaCaT) cells. Cells were exposed to UVB irradiation (0.1–1 kJ/m 2 ) either with or without specific inhibitors of NO [carboxy-2-pheryl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO)], extracellular signal-regulated kinase (ERK; U0126), c-Jun NH2-terminal kinase (JNK; SP600125), and p38 MAPK (SB203580). The levels of IL-10, TNF-α ,a nd NOwere then measured. The NO donor [(±)-N[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridinecarboxamide (NOR4)] was used to assess the involvement of NO in cytokine production. The activation of p38 MAPK was investigated in UVB-irradiated cells treated with p38 MAPK inhibitor, SB203580. The production of IL-10, TNF-α ,a nd NOby HaCaT cells increased upon exposure to UVB. The NO inhibitor, carboxy-PTIO suppressed NO production induced by UVB. NOR4 increased the production of TNF-α ,b utnot that of IL-10. The UVB-induced production of IL-10 and TNF-α were significantly suppressed by the specific inhibitors U0126, SP600125, and SB203580. In conclusion, UVB induced th ep roduction of proinflammatory mediators via activation of the p38 MAPK signaling pathway, suggesting that sunlight might promote the development of allergic diseases (such as dermatitis) through an augmented inflammatory response involving the increased production of proinflammatory cytokines and NO.

Expansion and commercialization of nanotechnology mean that it is important to understand the potential health hazards of manufactured nanoparticles. Here, we focused on the effect of fullerene, a type of nanoparticle already in commercial use, on delayed-type hypersensitivity (DTH) induced by methyl-bovine serum albumin (mBSA). Delayed-type hypersensitivity was induced with methyl-bovine serum albumin in female C57BL/6 mice. A colloidal suspension of crystalline C 60 (nano-C 60 ; average particle size 165 nm; 200 μL; 5.5 μg/mL) was injected intravenously twice, just before immunization and challenge with mBSA. Nano-C 60 treatment significantly attenuated footpad swelling, compared with that in DTH-disease control mice. Cytokine analysis indicated that nano-C 60 treatment switched the cytokine balance towards Th1-dominance. Pro-inflammatory cytokines IL-6 and IL-17 were significantly increased in DTH mice, and these increases were significantly suppressed by nano-C 60 treatment. Suppression of IL-17 by nano-C 60 was confirmed in an in vitro splenocyte culture. However, production of TNF-α was increased in DTH mice, and the increase was significantly enhanced by nano-C 60 treatment. The ratio of regulatory T (Treg) cells to total T (CD4 + ) cells was also significantly increased by nano-C 60 treatment, compared with that in DTH-disease control mice. Nano-C 60 treatment showed significant immunomodulatory effects in a mouse DTH model: IL-6 and IL-17 production was down-regulated, and the Treg cell ratio was up-regulated, concomitantly with attenuation of the pathology of DTH.

Gyokuheifusan (GHS) is a traditional Chinese medicine (TCM) formula used to treat various allergic diseases; however, there is little information about its clinical efficacy or mechanism of action. We previously showed that GHS down-regulates over-production of IgE and interleukin (IL)-4 in a mouse model of ovalbumin (OVA)-induced asthma, a major Th2-dominant disease. Here, we investigated the effect of GHS in an NC/Nga mouse model of atopic dermatitis (AD) induced by mite antigen. NC/Nga mice were immunized with an intradermal injection of mite antigen extract twice a week from 6 to 12 weeks of age. GHS (3.0 g/kg) was orally administered daily to the GHS-treated group for 6 weeks, and dermatitis score and total serum IgE level were measured sequentially. Spleen was harvested at 13 weeks of age, and the levels of cytokines released from splenocytes (interferon (IFN)-γ, IL-4 and IL-5) and IgG1/IgG2a levels in sera were measured by ELISA. Mice treated with GHS showed milder dermatitis than the disease-control group and the increase of total serum IgE level tended to be suppressed by GHS. In addition, GHS treatment suppressed the production of IL-4 and IgG1, though it did not affect the production of IFN-γ and IgG2a; i.e., GHS normalized the Th1/Th2 balance. These results suggest that GHS inhibits the development and reduces the severity of AD, at least in this model.

Diesel exhaust particles (DEP), a well-known air pollutant, exacerbate type I hypersensitivity conditions, such as asthma and pollen allergy. In this study, we examined the effect of diesel exhaust (DE) exposure on delayed-type hypersensitivity (DTH), a type IV hypersensitivity, induced with methyl-bovine serum albumin (mBSA) in C57BL/6 mice. Mice were exposed to DE containing DEP at a dose of 1.78 mg/m(3) in an inhalation chamber for 14 days. On Day 7, DTH mice and DE-exposed DTH mice were injected s.c. with 200 microl of 1.25 mg/ml mBSA emulsified with CFA in the dorsal region as initial sensitization. On Day 14, mice were injected s.c. into one footpad with 20 microl of 10 mg/ml mBSA dissolved in PBS as challenge. On Day15, footpad thickness and spleen weight were measured. Significant footpad swelling (%) was observed in DTH mice compared with normal control mice, and this swelling was significantly augmented by DE exposure. The levels of pro-inflammatory cytokines, including IFN-gamma, TNF-alpha, and IL-6, in DTH mice were significantly higher than in normal mice, and were also further enhanced by DE exposure. DE exposure increased production of IL-17, which enhances local tissue inflammation through up-regulation of pro-inflammatory cytokines, while production of IL-10, which inhibits local tissue inflammation through suppression of immune cell proliferation, was unchanged. No change was observed in the percentage of CD4(+)CD25(+)Foxp3(+)T regulatory (Treg) cells in splenic lymphocytes following DE exposure. IL-6 production was increased by DE, and this would facilitate the differentiation of naïve T cells to IL-17-producing Th17 cells, while concomitantly suppressing the competing differentiation pathway to IL-10-producing Treg cells. Our results indicate that DE inhalation may, in part, exacerbate the pathological symptoms of DTH and induction of pro-inflammatory cytokines such as IFN-gamma, TNF-alpha, IL-6 and IL-17.

We recently reported attenuation of autoimmune disease by low-dose gamma irradiation (repeated doses of 0.5 Gy) in MRL-lpr/ lpr mice. Here we studied the effect of low-dose gamma irradiation on experimental autoimmune encephalomyelitis (EAE), which is a rodent model of multiple sclerosis. SJL/J mice were immunized with myelin basic protein for the induction of EAE and were exposed to 0.5 Gy gamma rays once a week for 4 weeks. Radiation suppressed incidence and disease severity scores and delayed the onset of pathological changes. Pro-inflammatory cytokines (IFN-gamma and IL6), autoantibody and CD8+ cytotoxic T cells are involved in the appearance of EAE. Radiation suppressed the increases in these cytokines and the autoantibody production and blocked the increase in the population of CD8+ cytotoxic T cells. Production of IL17 by Th17 cells and/or gammadelta T cells, which plays a crucial role in EAE, was also significantly suppressed by radiation. Furthermore, a significant increase in regulatory T cells, which are involved in suppression of autoimmune disease, was found in irradiated EAE mice. These data suggest that low-dose gamma irradiation attenuates EAE through suppression of pro-inflammatory cytokines, reduction of cytotoxic T cells and induction of regulatory T cells.

Chlorinated derivatives of bisphenol A (ClBPAs) have been detected in wastewater from waste paper recycling plants. We previously reported that bisphenol A (BPA) and ClBPAs [3-chlorobisphenol A, 3,3'-dichlorobisphenol A, and 3,3',5-trichlorobisphenol A] irradiated with ultraviolet (UV) B or UVC (not with UVA) induced inhibition of cell growth, and that 3-hydroxybisphenol A (3-OHBPA) was detected in the photoproducts [Mutou, Y., Ibuki, Y., Terao, Y., Kojima, S., Goto, R., 2006b. Chemical change of chlorinated bisphenol A by ultraviolet irradiation and cytotoxicity of their products on Jurkat cells. Environmental Toxicology and Pharmacology, 21, 283-289]. The formation of hydroxylated BPAs by UV irradiation might contribute to the inhibition of cell growth, but the mechanism of the growth inhibition is not clarified. In this study, we investigated whether BPA and ClBPAs exposed to UVA, UVB, or UVC, and 3-OHBPA could induce the death of Jurkat cells and whether the pattern of cell death was apoptosis. ClBPAs exposed to UVB and UVC induced significant cell death, but those exposed to UVA and BPA did not. The cell death was apoptosis because chromatin condensation and DNA fragmentation were detected. Activation of caspase-3, -8, and -9 and cytochrome c release indicated that ClBPAs exposed to UVB or UVC induced apoptosis via typical apoptotic pathways. In addition, 3-OHBPA induced apoptosis similar to UVB- or UVC-irradiated ClBPA. These results suggested that the photoproducts of ClBPAs generated by UV irradiation, containing 3-OHBPA, contributed to the induction of apoptosis.

Tago, F., Tsukimoto, M., Nakatsukasa, H. and Kojima. S. Repeated 0.5 Gy Gamma Irradiation Attenuates Autoimmune Disease in MRL-lpr/lpr Mice with Suppression of CD3+CD4−CD8−B220+ T-Cell Proliferation and with Up-regulation of CD4+CD25+Foxp3+ Regulatory T Cells. Radiat. Res. 169, 59–66 (2008). MRL-lpr/lpr mice are used as a model of systemic lupus erythematosus. We previously reported attenuation of autoimmune disease in MRL-lpr/lpr mice by repeated γ irradiation (0.5 Gy each time). In this study, we investigated the mechanisms of this attenuation by measuring the weight of the spleen and the population of highly activated CD3+CD4−CD8−B220+ T cells, which are characteristically involved in autoimmune pathology in these mice. Splenomegaly and an increase in the percentage of CD3+CD4−CD8−B220+ T cells, which occur with aging in nonirradiated mice, were suppressed in irradiated mice. The high proliferation rate of CD3+CD4−CD8−B220+ T cells was suppressed in the irradiated animals. The production of au...

Core fucosylation and the bisecting N-acetylglucosamine residue are prominent natural substitutions of the N-glycan core. To address the issue of whether these two substituents can modulate ligand properties of complex-type biantennary N-glycans, we performed chemoenzymatic synthesis of the respective galactosylated and alpha2,3/6-sialylated N-glycans. Neoglycoproteins were then produced to determine these glycans' reactivities with sugar receptors in solid-phase assays and with tumor cells in vitro as well as their in vivo biodistribution profiles in mice. Slight protein-type-dependent changes were noted in lectin binding, including adhesion/growth-regulatory galectins as study objects, when the data were related to properties of N-glycans without or with only one core substituent. Monitoring binding in vitro revealed cell-type-dependent changes. They delimited the ligand activity of this glycan type from that of chains with un- and monosubstituted cores. A markedly prolonged serum half-life was conferred to the neoglycoprotein by the galactose-terminated N-glycan, which together with increased organ retention of all three neoglycoproteins underscores the conspicuous relevance for glycoengineering of pharmaproteins. The predominant presentation of the two branches in the disubstituted N-glycan as extended (alpha1,3-antenna) and backfolded (alpha1,6-antenna) forms, revealed by molecular dynamics simulations, can underlie the measured characteristics. These results obtained by a combined strategy further support the concept of viewing N-glycan core substitutions as non-random additions which exert a modulatory role on ligand properties. Moreover, our data inspire us to devise new, non-natural modifications to realize the full potential of glycoengineering for diagnostic and therapeutic purposes.

Oxidative stress is believed to greatly contribute to the pathogenesis of various diseases, including neurodegeneration. Impairment of mitochondrial energy production and increased mitochondrial oxidative damage are considered early pathological events that lead to neurodegeneration. Manganese superoxide dismutase (Mn-SOD, SOD2) is a mitochondrial antioxidant enzyme that converts toxic superoxide to hydrogen peroxide. To investigate the pathological role of mitochondrial oxidative stress in the central nervous system, we generated brain-specific SOD2-deficient mice (B-Sod2−/−) usingnestin-Cre-loxpsystem. B-Sod2−/−showed perinatal death, along with severe growth retardation. Interestingly, these mice exhibited spongiform neurodegeneration in motor cortex, hippocampus, and brainstem, accompanied by gliosis. In addition, the mutant mice had markedly decreased mitochondrial complex II activity, but not complex I or IV, in the brain based on enzyme histochemistry. Furthermore, brain lipid peroxidation was significantly increased in the B-Sod2−/−, without any compensatory alterations of the activities of other antioxidative enzymes, such as catalase or glutathione peroxidase. These results suggest that SOD2 protects the neural system from oxidative stress in the perinatal stage and is essential for infant survival and central neural function in mice.

Kupffer cells, which are resident macrophages in liver, can produce various cytokines and chemokines that induce hepatitis and liver fibrosis. It is suggested that extracellular ATP-induced activation of macrophage P2X7 receptor plays an important role in inflammation via release of pro-inflammatory mediators, but the role of P2X7 receptor in Kupffer cells remains unclear. Here, we show that activation of P2X7 receptor in Kupffer cells causes multiple inflammatory responses, using the clonal mouse Kupffer cell line (KUP5) that we previously established. Treatment of LPS-primed Kupffer cells with 3 mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of MAPK, cell lysis, IL-1β release, prostaglandin E2 (PGE2) release, high mobility group box1 (HMGB1) release, and major histocompatibility complex (MHC) class I shedding. These events were significantly suppressed by pretreatment with P2X7 antagonist A438079, indicating involvement of P2X7 receptor activation in these inflammatory responses. Our results suggest that extracellular ATP-induced activation of P2X7 receptor of Kupffer cells plays multiple roles in the inflammatory response in liver. P2X7 receptor might be a new therapeutic target for treatment of liver diseases.

We first examined the relation between the induction of glutathione and immune functions in mice after low-dose gamma-ray irradiation. Thereafter, inhibition of tumor growth by radiation was confirmed in Ehrlich solid tumor (EST)-bearing mice. The total glutathione level of the splenocytes transiently increased soon after irradiation and reached a maximum at around 4 h postirradiation. Thereafter, the level reverted to the 0 h value by 24 h postirradiation. A significantly high splenocyte proliferative response was also recognized 4 h postirradiation. Natural killer (NK) activity was also increased significantly in a similar manner. The time at which the response reached the maximum coincided well with that of maximum total glutathione levels of the splenocytes in the gamma-ray-irradiated mice. Reduced glutathione exogenously added to splenocytes obtained from normal mice enhanced the proliferative response and NK activity in a dose-dependent manner. The inhibitory effects of radiation on tumor growth was then examined in EST-bearing mice. Repeated low-dose irradiation (0.5 Gy, four times, before and within an early time after inoculation) significantly delayed the tumor growth. Finally, the effect of single low-dose (0.5 Gy), whole-body gamma-ray irradiation on immune balance was examined to elucidate the mechanism underlying the antitumor immunity. The percentage of B cells in blood lymphocytes was selectively decreased after radiation, concomitant with an increase in that of the helper T cell population. The IFN-gamma level in splenocyte culture prepared from EST-bearing mice was significantly increased 48 h after radiation, although the level of IL-4 was unchanged. IL-12 secretion from macrophages was also enhanced by radiation. These results suggest that low-dose gamma-rays induce Th1 polarization and enhance the activities of tumoricidal effector cells, leading to an inhibition of tumor growth.

The effect of 5,6,7,8-tetrahydrobiopterin (BH4), an endogenous antioxidant, on γ-ray-induced micronucleus frequency was examined in RAW264.7 cells. Exogenously added BH4 reduced the γ-ray-induced micronucleus frequency. Treatment with 1 mM sepiapterin, a biosynthetic precursor of BH4, also significantly reduced the micronucleus frequency induced by 3-10 Oy y-rays, by 20-35(/o at 1 8 h post-treatment. The reducing cffect of SP on γ-ray-induced micronucleus frequency was blocked by N-acetyl-5-hydroxytryptaminc and methotrexate, which are inhibitors of BH4 biosynthesis. These results suggest that intracellular BH4 biosynthesized from sepiapterin is involved in the reducing effect of SP on γ-ray-induced micronucleus frequency. To examine possible mechanisms underlying this action, changes of intracellular BH4 concentration after sepiapterin treatment were examined. Intracellular BH4 concentration peaked at 6 h and was maintained at a high level until 24 h after sepiapterin treatment. However, the γ-ray-induced micronucleus frequency at 6 h post-treatment with sepiapterin showed no change in comparison with that after irradiation alone. These results indicate that the reducing effect of BH4 on γ-ray-induced micronucleus frequency is not necessarily due to antioxidant activity, but may involve other mechanisms.

It has been reported that active oxygen and/or free radicals are produced in the central nervous system (CNS) compartment in patients with bacterial meningitis, so it is supposed that the levels of endogenous antioxidative scavengers in the cerebrospinal fluid (CSF) are elevated as an adaptive reaction to bacterial meningitis, which exerts severe stress on the human body. We assumed that they are also elevated in patients with convulsive diseases. Nitric oxide (NO) and endogenous antioxidative scavengers (glutathione (GSH), glutathione peroxidase (GPX), (total) superoxide dismutase (T-SOD), manganese superoxide dismutase (Mn-SOD), and catalase) were measured in CSF from a group of child patients with various neurological diseases and a control group. NO, GSH, and GPX activities in CSF from the patients with convulsive diseases were significantly higher than in those with aseptic meningitis or in the controls. Furthermore, all parameters in CSF from patients with bacterial meningitis were significantly higher than in any other group. The present study suggests that oxidative stress may be associated with the pathophysiology of convulsion and that its clinical attenuation will lead to improvement in the prognosis for convulsive diseases.

We first examined whether the increase of glutathione level induced by low-dose γ-ray irradiation is involved in the appearance of enhanced natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC), leading to delayed tumor growth in Ehrlich solid tumor (EST) -bearing mice. NK activity in ICR mouse splenocytes was significantly increased from 4h to 6h after a single whole-body γ-ray irradiation at 0.5 Gy, and thereafter decreased almost to the zero-time level by 24h post-irradiation. ADCC was also increased significantly in a similar way. Reduced glutathione exogenously added to splenocytes obtained from normal mice enhanced both NK activity and ADCC in a dose-dependent manner. The inhibitory effect of the radiation on tumor growth was then examined in EST-bearing mice. Repeated low-dose irradiation (0.5 Gy, four times, before and within an early time after the inoculation) significantly delayed the tumor growth.Finally, the effect of single low-dose (0.5 Gy), whole-body γ-ray irradiation on immune balance was examined in order to elucidate the mechanism underlying the anti-tumor immunity. The percentage of B cells in blood lymphocytes was selectively decreased after the radiation, concomitantly with an increase in that of helper T cell population. The IFN-γ level in splenocyte culture prepared from EST-bearing mice was significantly increased 48h after the radiation, though the level of IL-4 was unchanged. IL-12 secretion from macrophages was also enhanced by the radiation. These results suggest that low-dose γ-rays induce Th1 polarization and enhance the activities of tumoricidal effector cells, leading to a delay of tumor growth.

We previously showed that several small-dose 0.5 Gy whole-body γ-ray irradiation inhibits tumor growth in mice via elevation of the interferon (IFN)-γ/interleukin 4 (IL-4) ratio concomitantly with a decrease in the percentage of B cells. Here, we examined whether repeated small-dose (0.5 Gy, 10 times) γ-ray irradiation influences atopic dermatitis in an NC/Nga mouse model. It was found that repeated γ-ray irradiation increased total IgE in comparison with the disease-control group. Levels of IL-4 and IL-5 were increased versus the disease-control group, while IFN-γ was slightly decreased, resulting in a further decrease of the IFN-γ/IL-4 ratio compared with the disease-control group. These results indicate that repeated small-dose γ-ray irradiation may exacerbate atopic dermatitis. This may be because the irradiation induces not helper T lymphocyte 1 (Th1), but Th2 polarization in this atopic mouse model, i.e., the effects of small-dose irradiation may be different in conditions involving immune hypersensitivity and impaired immunity.

A series of enzymatic substitutions modifies the basic structure of complex-type biantennary N-glycans. Among them, a β1,2-linked N-acetylglucosamine residue is introduced to the central mannose moiety of the core-fucosylated oligosaccharide by N-acetylglucosaminyltransferase VII. This so-called LEC14 epitope can undergo galactosylation at the β1,2-linked N-acetylglucosamine residue. Guided by the hypothesis that structural modifications in the N-glycan alter its capacity to serve as ligand for lectins, we prepared a neoglycoprotein with the extended LEC14 N-glycan and tested its properties in three different assays. In order to allow comparison to previous results on other types of biantennary N-glycans the functionalization of the glycans for coupling and assay conditions were deliberately kept constant. Compared to the core-fucosylated N-glycan no significant change in affinity was seen when testing three galactoside-specific proteins. However, cell positivity in flow cytofluorimetry was enhanced in six of eight human tumor lines. Analysis of biodistribution in tumor-bearing mice revealed an increase of blood clearance by about 40%, yielding a favorable tumor/blood ratio. Thus, the extended LEC14 motif affects binding properties to cellular lectins on cell surfaces and organs when compared to the core-fucosylated biantennary N-glycan. The results argue in favor of the concept of viewing substitutions as molecular switches for lectin-binding affinity. Moreover, they have potential relevance for glycoengineering of reagents in tumor imaging.

The development of active targeting DDS nanoparticles is gaining attention in malignant tumor and other disease treatments that are seeking DDSs that do not produce side-effects. In the present work we have prepared two types of sugar-chain remodeled glycoprotein–liposome conjugates. These liposomal particles had a mean diameter of 100 nm, and maintained stable properties for several months. The lectin-binding affinity of 3′-sialyl-Lewis X-type sugar-chain-bearing liposomes was specific toward E-Selectin, and the dissociation constant was estimated to be in the level of about 300 pM. The uptake of 3′-sialyl-Lewis X-type sugar-chain-bearing liposomal nanoparticles was predominant for solid tumor tissue. These results emphasize the importance of structural features of sugar-chain ligands on the membrane surface of liposomal nanoparticles.

MRL-lpr/lpr mice, a model for various autoimmune diseases, were repeatedly irradiated with 0.5 Gy of gamma-rays, and changes in their autoimmune manifestations were investigated.MRL-lpr/lpr mice at 13 weeks of age were maintained in plastic cages and exposed whole-body to 0.5 Gy gamma-ray irradiation from a 137Cs source 5 times per week for 4 weeks, from the time they were 13 weeks old until they reached 17 weeks old. Changes of autoimmune manifestations were examined 3 weeks later at the 20th week.Splenomegaly, lymphadenopathy, and proteinuria in MRL-lpr/lpr mice were clearly ameliorated by a total dose of 10 Gy (0.5 Gy/day x 5 days/week for 4 weeks). Histologically severe disease-specific damage to the kidney and the salivary gland, i.e., glomerulonephritis and sialoadenitis, was also improved after irradiation. CD3+ CD4- CD8- CD45R/B220+ T cell numbers, which proliferate abnormally in MRL-lpr/lpr mice, were significantly decreased by the irradiation, possibly through induction of apoptosis. The elevated NO2- and NO3- (NO(x-) production by macrophages of MRL-lpr/lpr mice was lowered by the irradiation. The irradiation also prolonged the life span of MRL-lpr/lpr mice. These phenomena may contribute to the amelioration of autoimmune manifestations in MRL-lpr/lpr mice exposed to repeated small-doses of gamma-rays.Repeated small-dose gamma-ray exposure ameliorates the autoimmune manifestations in MRL-lpr/lpr model mice.

Human whole-blood was exposed to 137Cs gamma-rays or 50 keV/microm carbon ions in the presence or absence of glycine betaine, a beer component in vitro. The dicentrics of chromosome aberrations in human lymphocytes were significantly (p < 0.05) reduced by glycine betaine after irradiation with 4 Gy of either gamma-rays or carbon ions. The maximum protection by glycine betaine for gamma-rays or carbon ions was 37% and 20%, respectively. C3H/He female mice, aged 14 weeks, received an i.p. injection of glycine betaine 15 min before whole-body irradiation with gamma-rays or 50 keV/microm carbon ions. Glycine betaine significantly (p < 0.05) increased the percent survival of irradiated mice with either gamma-rays or carbon ions. In conclusion, glycine betaine is a potent protector against damages caused by low- and high-LET radiation.

Protein kinase C (PKC; also known as PRKC) is known to be an important participant in radiation-induced apoptosis. However, its role is not fully clarified. Using 3SBH5 cells, which are radiation-sensitive thymic lymphoma cells, the involvement and functions of PKC were assessed in radiation- induced apoptosis. PMA (phorbol 12-myristate 13-acetate), a PKC activator, inhibited the radiation-induced apoptosis in 3SBH5 cells. On the other hand, chelerythrine, a PKC inhibitor, potentiated apoptosis. In addition, Gö6976, a classical PKC (cPKC) inhibitor, which specifically inhibits PKC (alpha and betaI), also promoted apoptosis. Interestingly, post-treatment (20 min after irradiation) with Gö6976 had no effect on the radiation-induced apoptosis. These results suggest that cPKC is activated early after irradiation for anti-apoptosis signaling and contributes to the balance between cell survival and death. Indeed, an increase of cPKC activity involving PKC (alpha, betaI and betaII) was observed in the cytosolic fraction 3 min after irradiation with 0.5 Gy. However, no translocation of cPKC was observed in the cells after irradiation. Our findings indicate that activation of cPKC (alpha or beta) soon after irradiation is critical to the understanding of the regulation of radiation-induced apoptosis in radiation-sensitive cells.

We examined whether the increase of glutathione level induced by low dose gamma-ray irradiation is involved in the appearance of enhanced natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC), leading to delayed tumor growth in Ehrlich solid tumor-bearing mice. NK activity in ICR mouse splenocytes significantly increased from 4 h to 6 h after whole-body gamma-ray irradiation at 0.5 Gy, and thereafter decreased almost to the zero-time level by 24 h post-irradiation. ADCC also increased significantly in a similar way. Reduced glutathione exogenously added to splenocytes obtained from normal mice enhanced both NK activity and ADCC in a dose-dependent manner. Since immune functions were enhanced through the induction of cellular glutathione after low-dose irradiation, the inhibitory effect of the radiation on tumor growth was then examined in Ehrlich solid tumor-bearing mice. Tumor growth after inoculation was significantly delayed by the radiation. These results suggest that low-dose gamma-rays activate immune functions via an induction of glutathione, leading to a delay of tumor growth.

We have investigated the consequences of introducing a bisecting GlcNAc moiety into biantennary N-glycans. Computational analysis of glycan conformation with prolonged simulation periods in vacuo and in a solvent box revealed two main effects: backfolding of the α1–6 arm and stacking of the bisecting GlcNAc and the neighboring Man/GlcNAc residues of both antennae. Chemoenzymatic synthesis produced the bisecting biantennary decasaccharide N-glycan and its α2–3(6)-sialylated variants. They were conjugated to BSA to probe the ligand properties of N-glycans with bisecting GlcNAc. To assess affinity alterations in glycan binding to receptors, testing was performed with purified lectins, cultured cells, tissue sections and animals. The panel of lectins, including an adhesion/growth-regulatory galectin, revealed up to a sixfold difference in affinity constants for these neoglycoproteins relative to data on the unsubstituted glycans reported previously [Andre, S., Unverzagt, C., Kojima, S., Dong, X., Fink, C., Kayser, K. & Gabius, H.-J. (1997) Bioconjugate Chem. 8, 845–855]. The enhanced affinity for galectin-1 is in accord with the increased percentage of cell positivity in cytofluorimetric and histochemical analysis of carbohydrate-dependent binding of labeled neoglycoproteins to cultured tumor cells and routinely processed lung cancer sections. Intravenous injection of iodinated neoglycoproteins carrying galactose-terminated N-glycans into mice revealed the highest uptake in liver and spleen for the bisecting compound compared with the unsubstituted or core-fucosylated N-glycans. Thus, this substitution modulates ligand properties in interactions with lectins, a key finding of this report. Synthetic glycan tailoring provides a versatile approach to the preparation of newly substituted glycans with favorable ligand properties for medical applications.

Cerebrospinal fluid (CSF) neopterin been previously reported in various diseases. In this study CSF neopterin, biopterin, and nitrite/nitrate (NOx) Contents were measured and the correlation between them in child patients with various kinds of neurological diseases were investigated. Changes in the CSF neopterin levels in patients with bacterial meningitis were similar to those previously reported for those with bacterial meningitis; on the 2th hospital day they were significantly higher than on admission, and on the 6th hospital day they were tapered. The CSF biopterin levels and CSF NOx content in patients with bacterial meningitis on admission were significantly higher than those with other categories and were decreased gradually. Although patients with high levels of CSF neopterin tended to have high CSF biopterin levels in any categories, there was no significant correlation between CSF neopterin and biopterin levels. The CSF biopterin and NOx levels in patients with convulsions were higher than those with aseptic meningitis. Since the neuro-protective or anticonvulsant role for NO was previously reported, high CSF biopterin and NOx levels in patients having epilepsy or febrile convulsions may be regarded as one of the endogenous mechanisms for recovery from an overexcitatory brain in patients with convulsive diseases.

The effect of the intracellular 5,6,7,8-tetrahydrobiopterin (BH4) on the increased γ-glutamylcysteine synthetase (γ-GCS) mRNA expression induced by nitric oxide (NO) was investigated in RAW264.7 cells. Low doses of nitroprusside (SNP), ranging from 0.1 mM to 0.5 mM, significantly increased the intracellular glutathione levels along with the expression of mRNA for γ-GCS, a rate limiting enzyme of de novo glutathione synthesis. The increased expression was not abolished by exogenously added BH4 itself, but by sepiapterin, a precursor of BH4 synthesis, in a dose-dependent manner. The blockage by sepiapterin was ineffective in the presence of N-acetyl serotonin (NAS), an inhibitor of sepiapterin reductase. The increased γ-GCS mRNA expression was also inhibited by catalase, a scavenger of hydrogen peroxide. Evidence was further provided that BH4 effectively scavenged hydrogen peroxide. These results suggest that intracellular BH4 may play a role as an inhibitor of glutathione synthesis induced by NO via scavenging hydrogen peroxide, a mediator of the gene expression for the de novo glutathione synthesis pathway.

Because most of the diseases to which radon therapy is applied are related with activated oxygen, in this study, the effects of the radioactivity of radon and the thermal effects were compared under condition with the same chemical effects using the activity of superoxide dismutase (SOD), which is an oxidation inhibitor, and lipid peroxide and low-density lipoprotein (LDL)-cholesterol, which are closely involved in arteriosclerosis, as the parameters. The results were as follows, the SOD activity was significantly increased, and the lipid peroxide and LDL-cholesterol levels were significantly decreased on days 6 and 7. The results were about twofold larger in the radon group than in the thermo group. This suggests that the antioxidation function was more enhanced by radon therapy than by thermo therapy, and it was suggested that radon therapy may prevent the causes of lifestyle-related diseases such as arteriosclerosis. These findings are important in understanding the mechanism of diseases to which radon therapy can be performed, and most of which are called activated oxygen-related diseases.

The relation between induction of an increased glutathione level and the enhanced immune functions, including proliferative response and natural killer (NK) activity, of mouse splenocytes by a low dose of γ-rays was investigated. Glutathione level in mouse splenocytes significantly increased 2 h after whole-body γ-ray irradiation at 0.5 Gy, peaked at 4 h and thereafter, decreased almost to the initial (0 h) level within 12 h after irradiation. A significant enhancement of Con A-induced proliferation was recognized in the splenocytes from the whole-body-irradiated animals obtained at post-irradiation between 2 and 6 h. NK activity was also enhanced at the same time periods after the irradiation. Addition of glutathione to splenocytes obtained from normal mice enhanced both Con A-induced proliferative response and NK activity in a dose-dependent manner. These enhancements were completely blocked by buthionine sulfoximine, a specific inhibitor of the de novo pathway of glutathione synthesis. These results suggest that induction of endogenous glutathione synthesis in splenocytes after low-dose γ-ray irradiation is partially responsible for the appearance of enhanced immune function.

We have developed a suitable radiolabeling method for our new type of glycoprotein-liposome conjugate (GCL), in order to investigate its potential utility as a drug carrier that can target the cellular functions of carbohydrate-binding proteins. In order to obtain radiolabeled GCL with high labeling efficiency, we introduced p-hydroxyphenylpropyl groups into the liposome membrane through the amine moiety of a constitutive phospholipid, dipalmitoylphosphatidylethanolamine (DPPE) by using Bolton-Hunter reagent (BHR). Radioiodination of the introduced tyrosyl groups was performed by the Chloramine-T method. The labeling efficiency of the BHR-treated liposome conjugate was high in comparison with that of the BHR-untreated liposome conjugate. An in vitro inhibition study showed that the binding affinity of 125I-labeled BHR-treated GCL (125I-F3S-BH) with lectin was twice as high as that of untreated conjugate (125I-F3S). The biodistribution of 125I-F3S-BH in mice was considerably different from that of 125I-F3S. 125I-F3S-BH was more rapidly taken up by the liver and was more rapidly excreted from the liver than 125I-F3S. Moreover, 125I-F3S-BH accumulated more rapidly into the kidneys, which resulted a lower radioactivity in the blood circulation at an earlier time point than in the case of 125I-F3S. The characteristics of tumor accumulation of 125I-F3S-BH and 125I-F3S were similar to those in blood. If F3S is to be employed as an in vivo targeting ligand in biodistribution studies, BHR would be a suitable tool for radiolabeling because it allows GCL to retain the biological activity and characteristics of the unmodified conjugate.

The elevation of cellular glutathione (GSH) level induced by low concentrations of an nitric oxide (NO)-donor, sodium nitroprusside (SNP), and its effect on oxidant-induced cell injury were examined in RAW264.7 cells. The cellular GSH level increased 6 hr after exposure of the cells to SNP at low concentrations ranging from 0.1 to 0.5 mM, and the elevation followed the induction of mRNA coding for γ-glutamylcysteine synthetase, the ratelimiting enzyme of the de novo glutathione synthesis pathway. Pre-treatment of cells with low concentration of SNP (less than 0.25 mM) at 12 hr prior to exposure to menadione (MEND), an superoxide anion (O2 ‐ )-donor, significantly suppressed the cell injury induced by MEND alone. Simultaneous treatment with a higher concentration of SNP (1.0 mM or more) also blunted the MEND-induced cell injury. Low and high doses of NO both seem to show a preventive effect against oxidant injury: NO may protect against oxidant injury by up-regulating GSH synthesis at low concentrations, while at high concentrations it may directly react with radical oxygen species (ROS), thus acting as a free radical scavenger and blunting oxidant injury. These results suggest that modulation of the cellular glutathione metabolism through intracellular NO is a potential mechanism for enhancing the antioxidant defense of cells.

There is considerable evidence from experimental studies in animals, as well as from clinical reports, that low-dose radiation hormesis is effective for the treatment of cancer and ulcerative colitis. In this study, we present 3 case reports that support the clinical efficacy of low-dose radiation hormesis in patients with these diseases. First, a patient with prostate cancer who had undergone surgical resection showed a subsequent increase in prostate-specific antigen (PSA). His PSA value started decreasing immediately after the start of repeated low-dose X-ray irradiation treatment and remained low thereafter. Second, a patient with prostate cancer with bone metastasis was treated with repeated low-dose X-ray irradiation. His PSA level decreased to nearly normal within 3 months after starting the treatment and remained at the low level after the end of hormesis treatment. His bone metastasis almost completely disappeared. Third, a patient with ulcerative colitis showed a slow initial response to repeated low-dose irradiation treatment using various modalities, including drinking radon-containing water, but within 8 months, his swelling and bleeding had completely disappeared. After 1 year, the number of bowel movements had become normal. Interest in the use of radiation hormesis in clinical practice is increasing, and we hope that these case reports will encourage further clinical investigations.

We previously reported that low doses (0.25-0.5 Gy) of γ-rays induce intracellular antioxidant, radioresistant, DNA damage repair, and so on. Meanwhile, we have recently reported that ATP is released from the cells exposed to low-dose γ-rays. Here, it was investigated whether or not γ-radiation-induced release of extracellular ATP contributes to various radiation effects, in paricular, focusing on the inductions of intracellular antioxidant and DNA damage repair. Irradiation with γ-rays or exogenously added ATP increased expression of intracellular antioxidants such as thioredoxin and the increases were blocked by pretreatment with an ecto-nucleotidase in both cases. Moreover, release of ATP and autocrine/paracrine positive feedback through P2Y receptors serve to amplify the cellular repair response to radiation-induced DNA damage. To sum up, it would be suggested that ATP signaling is important for the effective induction of radiation stress response, such as protection of the body from the radiation and DNA damage repair. In addition, the possibility that this signaling is involved in the radiation resistance of cancer cells and beneficial effect on the organism of low-dose radiation and radiation adaptive response, would be further suggested.

Resident macrophages in the liver (Kupffer cells) produce various cytokines and chemokines, and have important roles in hepatitis and liver fibrosis. The cells are activated by various factors, for example lipopolysaccharide (LPS), which is an endotoxin and is high in the blood of patients with liver cirrhosis. Involvement of P2 receptors in the release of pro-inflammatory cytokines from Kupffer cells is little. In this study, we investigated purinergic signaling in the release of pro-inflammatory cytokines, such as IL-6 and TNF-α, from liver Kupffer cells of C57BL/6 mice (KUP5 cells). KUP5cells were isolated from C57BL/6 mice and cultivated with Dulbecco's modified Eagle's medium. The cells were stimulated with LPS. LPS-induced IL-6 production by KUP5 cells was suppressed significantly by pretreatments with non-selective P2 antagonist suramin, P2Y13antagonist MRS2211, and ecto-nucleotidase, whereas P2Y receptor agonists, significantly increased the IL-6 production. P2Y13knockdown reduced LPS-induced IL-6 production, but by less than 50%. These results would suggest that P2Y receptors including P2Y13and others, may involves in LPS-induced IL-6 production in Kupffer cells, leading to the liver inflammation. Therefore, we first showed the importance of purinergic signaling via P2Y receptors in the activation of Kupffer cells and liver injury, which is worthwhile in drug development for liver diseases.

Regulatory T cells (Treg) play a role in suppression of immune response, including anti-tumor immunity. We have recently reported that treatment of naïve CD4 T cells with adenosine A(2B) receptor antagonist PSB603 under Treg-skewing conditions inhibits expression of Foxp3, a marker of differentiation to Treg, without blocking IL-2 production or CD25 expression, which are activation markers, in CD4 T cells. We hypothesized that PSB603 suppresses cancer growth and metastasis by inhibiting induction of Treg, thereby facilitating anti-tumor immunity. In this study, we first examined the effect of PSB603 on tumor growth in B16 melanoma-bearing C57BL/6 mice. Administration of PSB603 significantly suppressed the increase of tumor volume as well as the increase of Treg population in these mice. The populations of CD4 and CD8 T cells were higher and splenic lymphocyte-mediated cytotoxicity towards B16 melanoma was significantly increased in PSB603-treated mice. We confirmed that PSB603 did not reduce the viability of B16 melanoma cells in vitro. Moreover, we also examined the effect of PSB603 on tumor metastasis in pulmonary metastasis model mice intravenously injected with B16 melanoma cells. The metastasis was also suppressed in PSB603-treated mice, in which the population of Treg was significantly lower. Overall, our results suggest that A(2B) receptor antagonist PSB603 enhances anti-tumor immunity by inhibiting differentiation to Treg, resulting in a delay of tumor growth and a suppression of metastasis.

There is extensive evidence that nanoparticles (NPs) cause adverse effects in multiple organs, including liver, though the mechanisms involved remain to be fully established. Kupffer cells are macrophages resident in the liver, and play important roles in liver inflammation induced by various toxic agents, including lipopolysaccharide (LPS). Interleukin-1 (IL-1) family members IL-1α,β are released from LPS-primed macrophages exposed to NPs, including silica NPs (SNPs), via activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes. Here, we investigated the mechanism of production of IL-1β via activation of inflammasomes in mouse Kupffer cell line KUP5, focusing on the role of purinergic signaling via P2X7 receptor. IL-1β production by LPS-primed KUP5 cells exposed to SNPs was increased dose-dependently, and was greatest in response to SNPs with a diameter of 30 nm (SNP30), as compared with 70-nm and 300-nm SNPs (SNP70 and SNP300). ATP release was also highest in cells exposed to SNP30. Treatment of LPS-primed KUP5 cells with ATP also induced a high level of IL-1β production, similar to that induced by SNP30. IL-1β production was significantly inhibited by apyrase (an ecto-nucleotidase) and A438079 (a P2X7 antagonist/ATP-release inhibitor). Production of reactive oxygen species (ROS) was confirmed in cells exposed to SNP30. In conclusion, ATP released from P2X7 receptor in response to stimulation of KUP5 cells with SNP30 induces ROS production via cell-membrane NADPH oxidase. The ROS causes activation of inflammasomes, leading to caspase-1-dependent processing of IL-1β.

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC.

We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant H2AX (γH2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y6 and P2Y12 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y6 receptor is highly expressed in A549 cells, but P2Y12 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y6 receptor antagonist MRS2578 markedly reduced the survival rate of γ-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y6 and P2Y12 receptors is involved in the repair response to γ-ray-induced DNA damage, we next examined whether γ-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to γ-ray-induced DNA damage.

We have investigated the effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transport function in brain slices using gas–tissue autoradiography technique with [ 15 O]O 2 . Brain slices were preincubated in Krebs-Ringer phosphate medium bubbled with 100% O 2 for 30 min at 37°C. (1) Control culture was incubated in the same medium bubbled with 100% O 2 for 5–40 min at 37°C, then for another 30 min under the same conditions. (2) In vitro ischemia was induced by placing the culture in the medium deprived of glucose and bubbled with 100% N 2 for 5–40 min, then returning it to control conditions and culturing for another 30 min. (3) In vitro hypoxia was induced by placing the culture in the medium with glucose and bubbled with 100% N 2 for 5–40 min, then returning it to the control conditions for 30 min. After the three different treatments, the [ 15 O]O 2 fixation by brain slices reflect to mitochondrial electron transport function was determined using gas–tissue autoradiography technique with [ 15 O]O 2 . The fixation of [ 15 O]O 2 by striatum, cerebral cortex and hippocampus was reduced dependent upon the period of in vitro ischemic treatment. In contrast, the [ 15 O]O 2 fixation by those brain regions was only slightly reduced by hypoxia treatment. The reduction in [ 15 O]O 2 fixation induced by ischemic treatment was prevented by an antioxidant: glutathione, glutathione monoethyl ester or acetylsalicylic acid. The preventive effect of antioxidants on the mitochondrial damage induced by ischemia was more remarkable in the striatum than in the cerebral cortex and hippocampus. In the comparison of [ 15 O]O 2 fixation between ischemia-treated young and senescent brain slices, reduction of 15 O fixation by every brain region examined was more prominent in senescence than in the young. These results suggest that gas–tissue autoradiography using [ 15 O]O 2 is useful to assess mitochondrial electron transport dysfunction induced by ischemia treatment in brain slices and that the oxidative stress participates in the mechanism of ischemia-induced dysfunction in mitochondria.

To examine the relation between the induction of an increased glutathione level and the elevated proliferative response of mouse splenocytes by a small dose of gamma-rays.Male ICR strain mice, 7 weeks of age, were divided into irradiated and non-irradiated control groups. Irradiation was done with gamma-rays from a 137Cs source at a dose of 50 cGy (1.11 Gy/min). Glutathione content in the splenocytes was measured using a modified spectrophotometric technique. Concanavalin A (Con A)-induced proliferative response of the splenocytes after whole-body gamma-ray irradiation was estimated from the 3H-thymidine incorporation into the cells.The glutathione level in mouse splenocytes increased 2 h after whole-body y-ray irradiation at 50cGy, peaked at 4h and thereafter decreased almost to the zero-time level by 12-h postirradiation. A significant enhancement of Con A-induced proliferation was observed in the splenocytes obtained from the whole-body-irradiated animals between 2h and 6h post-irradiation. Glutathione exogenously added to splenocytes obtained from normal mice enhanced the Con A-induced proliferation of splenocytes in a dose-dependent manner. This enhancement was completely blocked by buthionine sulfoximine, a specific inhibitor of the de novo pathway of glutathione synthesis.The induction of endogenous glutathione immediately after low-dose gamma-ray irradiation is at least partially responsible for the enhancement of immune function, and may throw light on the mechanisms of carcinostatic effects induced by low dose ionizing radiation.

We studied the effects of a single post whole-body low-dose irradiation (50 cGy of gamma-ray) on mice with ferric nitrilotriacetate (Fe3+-NTA)-induced transient hepatopathy. As a result, low-dose irradiation accelerated the rate of recovery. Based on the changes in glutamic oxaloacetic transaminase (GOT) activities, glutamic pyruvic transaminase (GPT) activities and lipid peroxide levels, it was shown that hepatopathy was improved by low-dose irradiation 3 h after Fe3+-NTA administration. This may be because of the enhancement of antioxidant agents such as total glutathione (GSH + GSSG), glutathione peroxidase (GPX), glutathione reductase (GR) and gamma-glutamylcysteine synthetase (gamma-GCS) by low-dose irradiation. These findings suggest that low-dose irradiation relieved functional disorders at least in the livers of mice with active oxygen species related diseases.

To investigate the mechanism of the intracellular glutathione elevation induced by low-dose gamma-radiation.RAW 264.7 cells were irradiated with 1-400cGy gamma-rays. Intracellular total glutathione content was determined by DTNB-recycling assay. Expression of mRNA for intracellular glutathione synthesis-related enzymes with or without treatment with various inhibitors of second messengers of gene expression were examined by Northern blot analysis.Expression of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme of the de novo glutathione synthesis pathway, was elevated much more than that of glutathione reductase (GR) mRNA after exposure to 50cGy gamma-rays. The low-dose gamma-ray-induced gamma-GCS mRNA elevation was abolished by inhibitors of protein kinase C and protein tyrosine kinase, as well as by the calcium ion channel blocker, nifedipine. Calcium-related reagents, such BAPTA/AM and EGTA, chelators of intra- and extracellular Ca2+ respectively, and a Ca2+ ionophore (A23187), also strongly blocked the elevation of gamma-GCS mRNA expression induced by gamma-rays.The increase of intracellular glutathione in RAW 264.7 soon after low-dose gamma-ray exposure mainly occurs through the operation of the de novo pathway, following by the induction of gamma-GCS mRNA, for which elevation of intracellular calcium is required.

Summary It has been previously reported that 5,6,7,S-tetrahydrobiopterin (BH4) modulates HL-60 cell death induced by a nitric oxide (NO) donor, S-nitroso-N-acetyl-D, L-penicillamine (SNAP). In this study, the role of endogenous BH4 was investigated in lipopolysaccharide (LPS)-induced apoptotic cell death. LPS induced an increase of DNA fragmentation and of nitrite and nitrate content (NOx content) in the macrophage- like RAW 264.7 cell line. 2,4-Diamino-6-hydroxypyrimidine (DAHP) , an inhibitor of BH4 synthesis, suppressed both of them. The NOx content of cells treated with LPS and interferon-y was much higher than that of cells treated with LPS alone. However, the degree of apoptotic cell death induced by LPS and interferon-y did not differ significantly from that induced by LPS alone. Further investigation revealed that LPS-induced cell death of RAW264.7 cells was mainly mediated by reactive oxygen species, such as hydrogen peroxide (H2O2). From these data, it was speculated that BH4 might be a modulator in NO-induced apoptosis, in which BH4 involves LPS-induced cell death by its function asa cofactor of inducible NO synthase and it suppresses the cell death mediated by NO and/or H2O2 via an antioxidative activity.

Protein (lectin)-carbohydrate (cellular glycoconjugate) recognition is operative in biochemical information transfer. Galectins constitute a family of endogenous galactoside-binding lectins with conserved features in the binding site. The members of this lectin category are assumed to be involved in cell adhesion and growth regulation. To assess to what extent the different modes of binding-site presentation and/or carbohydrate fine-specificities will affect aspects of galectin behavior, homodimeric cross-linking galectin-1 and monomeric chimeric galectin-3, with its collagenase-sensitive stalk linked to the carbohydrate-recognition domain, were investigated. Cell-surface expression of the two galectins and accessible galectin-binding sites on various tumor cell lines was ascertained by FACScan analysis. In particular, ligand accessibility for the two galectins differed for the tested cell line types. Binding of tumor cells to laminin and plasma or placental fibronectin was generally reduced by treatment of cells or matrix with galectins. Galectin-3 was more efficient than galectin 1 at impairing laminin's potency as matrix. Cell binding of galectin-1, on the other hand, proved on average more effective for blocking cell association to fibronectins after its preincubation with cell suspensions. Differences were also apparent in the biodistribution of the galectins, where an avian homolog of galectin-1 served as the control to distinguish effects of spatial and sugar-binding features. Histopathological analysis of lymph-node-negative and -positive breast and colorectal carcinomas (n = 180 including 60 metastatic lesions) indicated a correlation of either increased galectin-1 binding and reduced galectin-3 expression or reduced binding of both galectins with the occurrence of lymph node lesions. Together with data on the heparin-binding lectin, revealing reduced expression to be associated with a positive lymph-node status in the breast cancer group, these results can be interpreted to reflect cell-type-dependent requirements of galectin ligand presentation during the metastatic cascade. By introducing mammalian lectins to lectin-histochemical studies, the detection of quantitative differences in glycosylation brings an understanding of its cell biological significance one step closer.

Bacteria and mammalian cells show an adaptive response to oxidative stress. Pre-treatment with small amounts of oxidant induces resistance to the subsequent, otherwise lethal, doses of oxidant. Previous studies have shown that this adaptive response involves the induction of superoxide dismutase (SOD), glutathione peroxidase (GPX), metallothionein, heat shock proteins, and other factors. The toxicity of ionizing radiation, particularly at low doses, to living cells, is thought to be due to reactive oxygen species (ROS) formation. Thus, adaptive responses to low doses of ionizing radiation and to oxidants are likely to be similar. In this paper, the induction of endogenous glutathione (GSH) together with antioxidant enzymes induced by low-dose radiation was reviewed. Futhermore, the applicable possibility of this efficacy for the prevention of and / or therapy of various reactive oxygen species (ROS)-related diseases including Parkinson's diseases, aging, and diabetes, was discussed.