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Flk1-expressing (+) mesodermal cells are useful source for the generation of hematopoietic cells and cardiomyocytes from pluripotent stem cells (PSCs). However, they have been reported as a heterogenous population that includes hematopoietic and cardiac progenitors. Therefore, to provide a method for a highly efficient production of hematopoietic cells and cardiomyocytes, cell surface markers are often used for separating these progenitors in Flk1(+) cells. Our recent study has shown that the expression of coxsackievirus and adenovirus receptor (CAR), a tight junction component molecule, could divide mouse and human PSC- and mouse embryo-derived Flk1(+) cells into Flk1(+)CAR(-) and Flk1(+)CAR(+) cells. Flk1(+)CAR(-) and Flk1(+)CAR(+) cells efficiently differentiated into hematopoietic cells and cardiomyocytes, respectively. These results indicate that CAR is a novel cell surface marker for separating PSC-derived Flk1(+) mesodermal cells into hematopoietic and cardiac progenitors. We herein describe a differentiation method from PSCs into hematopoietic cells and cardiomyocytes based on CAR expression.

CD25+ regulatory T (T reg) cells play a suppressive role in experimental autoimmune uveoretinitis as well as experimental airway inflammation but their involvement in the development of allergic conjunctivitis (AC) remains unclear. We therefore investigated whether T reg cells play a role in the development of experimental AC (EC). BALB/c and C57BL/6 mice were actively immunized with ragweed (RW). The mice were treated with an anti-CD25 Ab (PC61) or control normal rat IgG (nrIgG) either 2 days prior to active immunization or during the induction phase (days 0, 2, 4, 6 and 8). Ten days after active immunization, the mice were challenged with RW-containing drops. Twenty-four hours after the challenge, the conjunctivas were harvested for histological analysis of eosinophil infiltration, and the spleens were harvested for cell culture for splenocyte transfer. Cultured splenocytes were transferred into syngeneic mice, and 4 days after the transfer, the recipient mice were challenged with RW. Twenty-four hours after the challenge, conjunctivas were collected for histological analysis. Pretreatment with PC61 did not affect EC in either strain of mice; however, treatment with PC61 during the induction phase significantly suppressed EC in C57BL/6 mice. In contrast, transfer of RW-primed splenocytes from mice treated with PC61 induced EC that was significantly more severe regardless of strain and treatment protocol. The finding that T reg cells play a suppressive role in the development of EC in splenocyte transfer experiments suggests that modulation of T reg cells may be a possible therapy for AC.

Although CD4+ Th2 cells clearly play an essential role in the development of experimental allergic diseases, the functions CD8+ T cells may have in these diseases have been investigated less extensively and remain controversial. Here, we investigated the roles of CD8+ T cells in the development of experimental allergic conjunctivitis (EC). EC was induced in CD8alpha-deficient (CD8KO) mice and wild-type (WT) mice by active immunization with short ragweed pollen (RW) followed by challenge with RW-containing eye drops. Alternatively, EC was induced by transferring RW-primed splenocytes followed by RW challenge. With regard to actively immunized mice, CD8KO mice showed significantly less severe eosinophil infiltration of the conjunctiva and lower total IgE levels, although the levels of the other Igs were equivalent between the two strains. Cytokine production by cultured splenocytes also did not differ, but the WT conjunctivas showed upregulated IL-5 and IL-6 expression and greater upregulation of IL-4 expression than the conjunctivas of CD8KO mice. Thus, CD8+ T cells may play a significant role during the induction phase by aiding IgE production and the generation of Th2 cytokines in the conjunctiva, thus promoting the development of EC. In contrast, splenocytes from CD8KO mice induced significantly more severe EC in WT mice than cells from WT mice. In addition, transfer of RW-primed splenocytes induced significantly more severe eosinophil infiltration in CD8KO recipient mice. Thus, CD8+ T cells promote the development of EC during the induction phase, but suppress it during the effector phase.

We have reported that natriuretic effects of K(+) are involved in enhancement of renal kallikrein-kinin system. The study was aimed to examine 1) comparison of augmentative effects of K(+) on urinary KK excretion with non-specific washout effects by trichlormethiazide (thiazide), polyethyleneglycol 200 (PEG) and rapid physiological saline infusion, 2) contribution of Ca(2+) on the K(+)-induced increase in renal kallikrein secretion. Renal kallikrein activities were measured as fluorescence activities of methylcoumarinylamide-labeled synthetic substrate of tissue kallikrein (TK). Increases in urinary TK excretion were simultaneously observed with diuresis caused by thiazide, PEG, and rapid saline infusion. K(+) infusion increased urinary TK excretion with a diuretic response same as the control. K(+), but not thiazide, showed an early increase in renal TK secretion dose dependently in the kidney slices. Increases in renal TK secretion persisted during treatment with K(+). Neither voltage-dependent Ca(2+)-channel blockers such as verapamil and nifedipine nor simultaneous treatment of EDTA affected on the K(+)-induced increase in renal TK secretion. While, EDTA decreased the K(+)-induced increases in renal TK secretion with time. Caffeine also had an early effect on the increase in renal TK secretion. K(+)-induced increases in renal TK secretion was demonstrated even after treatment with ryanodine or depletion of caffeine-sensitive intracellular Ca(2+) by thapsigargin. It was indicated that the increase in renal TK secretion by K(+) depends on the intracellular Ca(2+) and the caffeine-sensitive release of intracellular Ca(2+) may not be involved in this response. Mechanisms for the K(+)-induced increase in renal TK secretion needs to be further elucidated.

DNA topoisomerase II is a nuclear enzyme that regulates DNA topology via transient double-strand breaks in the DNA helix (5, 38, 39). Topoisomerase II is involved in cell proliferation and has been implicated in indispensable cellular processes, such as replication, transcription, recombination, and chromosomal condensation and segregation (38). This enzyme also has an essential function as a structural component of mitotic chromosome and interphase nuclear scaffolds (9, 14). Given the fact that expression of topoisomerase II in proliferating cells is higher than that in quiescent cells, this enzyme is a clinically useful target to elicit cytotoxic effects in proliferating tumor cells. Indeed, a variety of anticancer agents target topoisomerase II (20) and interfere with its catalytic activity by trapping the enzyme in a form that is covalently bound to DNA. These stable enzyme-associated DNA complexes induce DNA damage and cell death. In addition to catalytically inactivating topoisomerase II, previous studies have shown that aberrant expression of topoisomerase II is associated with the induction of apoptosis (28, 37). While forced expression of topoisomerase IIα in cells triggered apoptotic cell death, nuclear localization of the enzyme was required for efficient apoptotic induction. By contrast, another report demonstrated that depletion of topoisomerase IIα conferred induction of apoptosis (1). Taken together, these results suggest that appropriate regulation of topoisomerase IIα expression is essential for cell viability and proliferation. In this regard, deregulated expression of topoisomerase IIα is associated with the commitment of apoptotic cell death; however, the mechanism remains unclear. The protein kinase C (PKC) family of serine/threonine kinases is subdivided into (i) conventional PKCs (PKC α [PKCα], PKCβ, and PKCγ) that are calcium dependent and activated by diacylglycerol (DAG), (ii) novel PKCs (PKCδ, PKCɛ, PKCθ, and PKCμ) that are calcium independent and activated by DAG, and (iii) atypical PKCs (PKCζ and PKCλ) that are calcium independent and not activated by DAG (32). The ubiquitously expressed novel PKC, PKCδ, is tyrosine phosphorylated and activated by c-Abl and Lyn in the response to DNA damage (45, 48). PKCδ interacts with the nuclear DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (2). Phosphorylation of DNA-PKcs by PKCδ inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate downstream targets (2). In addition, cells deficient in DNA-PK are resistant to apoptosis induced by overexpression of the PKCδ catalytic domain. Other studies have demonstrated that the nuclear complex of c-Abl and Lyn includes the protein tyrosine phosphatase SHPTP1 (Src homology 2 domain [SH2]-containing protein tyrosine phosphatase 1) (21, 42) and that PKCδ phosphorylates and inactivates SHPTP1 in response to DNA damage (44). In cells that respond to genotoxic stress with apoptosis, PKCδ is cleaved by caspase-3 into a constitutively active catalytic fragment (PKCδCF) (10, 11). The finding that PKCδCF induced nuclear condensation and DNA fragmentation indicates that cleavage of PKCδ contributes to the apoptotic response (15). In this context, a recent study has demonstrated that PKCδ translocated to the nucleus and regulated Rad9 by phosphorylation in the apoptotic response to DNA damage (46). Furthermore, another study showed that cells derived from PKCδ-null transgenic mice were defective in mitochondrion-dependent apoptosis induced by various agents such as UV irradiation and hydrogen peroxide (27). These findings collectively support an essential role for PKCδ in the induction of apoptosis in the genotoxic stress response. The present study demonstrates that PKCδ interacts with topoisomerase IIα. This interaction is required for stabilization and activation of topoisomerase IIα following DNA damage. The results also demonstrate that genotoxic stress-induced topoisomerase IIα activation confers the induction of PKCδ-mediated apoptosis.

Balb/c and C57BL/6 mice have been reported to be biased towards Th2 and Th1 immune responses, respectively. We investigated which strain is more susceptible to the development of experimental immune-mediated blepharoconjunctivitis (EC), which is predominantly mediated by Th2 immune responses. EC was induced by three different methods in Balb/c and C57BL/6 mice using ragweed (RW) as the antigen. The mice were thus either actively immunized with RW, passively immunized by transfer of RW-primed T cells, or passively immunized by transfer of RW-specific IgE, followed by RW challenge in eye drops. Twenty-four hours after the challenge, conjunctivas, sera and spleens were harvested for histological analysis, measurement of serum IgE and assessment of cellular immune responses, respectively. The responses of the Balb/c and C57BL/6 mice were compared. In addition, to assess the involvement of IFN-gamma in the development of EC in the two strains, IFN-gamma knockout (GKO) mice of the two strains were actively immunized and evaluated as above. Regardless of the method of induction, EC, as determined by the degree of eosinophil infiltration into the conjunctiva, was more severe in Balb/c mice than in C57BL/6 mice. Moreover, more IgE was produced by actively immunized Balb/c mice than C57BL/6 mice and RW-primed splenocytes from Balb/c mice produced more IL-4 but less IFN-gamma than those from C57BL/6 mice. EC could be induced in the GKO mice of both strains. However, when their EC was compared to that in WT mice, significantly less infiltration of eosinophils was noted in the Balb/c GKO mice. Taken together, Balb/c mice are more susceptible to EC than C57BL/6 mice and this higher susceptibility might be related to the Th2 immune response bias of Balb/c mice. Furthermore, the involvement of endogenous IFN-gamma in the development of EC in these two strains differs.

Changes in the radical-scavenging activities and the total phenol content of sixteen spices (clove, allspice, cinnamon, nutmeg, mustard, cumin, ginger, fennel, fenugreek, black pepper, red pepper, mace, coriander, turmeric, cardamom and white pepper) were determined for different heating times (1, 3 and 6h) at 100°C. Most of the spices showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity (4-1353μmol Trolox eq./g), peroxy radical-scavenging activity (31-1019μmol Trolox eq./g), and total phenol content (5-1267μmol gallic acid eq./g). Clove was found to have the highest radical-scavenging activity followed by allspice and cinnamon. After heating, both DPPH and peroxy radical-scavenging activities as well as the total phenol content increased in most of the spices. A distinct increase in the activities was found in some spices such as black pepper, red pepper and turmeric. A high correlation coefficient was found between the total phenol content and peroxy radical-scavenging activity.

Innate immune responses that operate through Toll-like receptors (TLRs) are actively involved in the development of diseases predominantly mediated by adaptive immune responses. This is true also for allergic disease, as TLRs have been found to be involved in the development of allergic airway inflammation. We investigated whether stimulating TLR2 also abrogates murine allergic conjunctivitis by upregulating Th1 responses. We found that treating mice during the efferent phase with the TLR2 agonist Pam3CSK4 significantly suppressed eosinophil infiltration into the conjunctiva. However, Pam3CSK4 treatment inhibited both the Th1 and Th2 responses in the mice, and also suppressed eosinophil infiltration in IFN-gamma knockout mice. Flow cytometric analysis demonstrated that Pam3CSK4 treatment significantly elevated the numbers of annexin V-positive splenocytes, especially CD4 positive T cells. Thus, the stimulation of TLR2 during the efferent phase of murine allergic conjunctivitis suppresses eosinophil infiltration by inducing CD4 positive T-cell apoptosis rather than upregulating Th1 responses.

L-Ascorbic acid (AsA) has numerous biological activities. It is known that AsA is unstable under neutral and alkaline conditions, degrading almost completely within several hours, whereas it is relatively stable under acidic conditions. The present study investigated the effect of grape seed extract (GSE), which contains proanthocyanidins, on the stability of AsA under neutral and alkaline conditions. The addition of GSE to AsA solution in 3,3′-dimethylglutaric acid-tris(hydroxymethyl)aminomethane-2-amino-2-methyl-1,3-propanediol (GTA) (50mM, pH 7.0 or 10.0) buffer significantly increased the remaining amount of AsA and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, compared with those of AsA alone. In particular, it was clarified that GSE contributed to the stability of AsA at an alkaline pH. We also investigated the effect of GSE on the stability of AsA under quasi-physiological conditions. It was revealed that GSE stabilized AsA in simulated intestinal juice (pH 8.5) at 37°C. DPPH radical-scavenging activity was closely correlated with the remaining amounts of AsA. The present results, although not directly transferable to in vivo conditions, suggest that GSE may stabilize AsA under neutral and alkaline conditions and affect the physiological activity of AsA.

The 4-1BB receptor acts as a costimulator in CD8+ T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-γ production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-γ knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-γ.

Background: Studies with interferon-γ knockout (GKO) mice showed that endogenous IFN-γ suppresses the infiltration of inflammatory cells into the conjunctiva. To examine whether this phenomenon is universally true, we induced conjunctival inflammation by four different immunization protocols. Methods: Both wild type (WT) and GKO mice (C57BL/6 background) were immunized with ragweed (RW) in aluminum hydroxide (alum). Two different immunization protocols were used: either the emulsion was injected into only the left hind footpad or it was also injected into the tail base (50 μg RW in 2 mg alum per injection site). In addition, to compare the effects of the immunization dose of RW and the immunization site, 100 μg RW in 2 mg alum was injected into only the left hind footpad and 25 μg RW in 2 mg alum per injection site was injected into both the left hind footpad and the tail base. Ten days later, the mice were challenged with 2 mg RW in 10 μl PBS. Twenty-four hours later, the conjunctivas were analyzed histologically, and the cellular and humoral immune responses in the spleens and sera were determined, respectively. Results: Similar to a previous report, GKO mice showed significant eosinophilic infiltration into the conjunctiva after the footpad only injection of 50 μg RW. However, injection of 50 μg RW per injection site into the footpad plus the tail base resulted in comparable levels of eosinophilic infiltration in WT and GKO mice. On the contrary, either immunization of 100 μg RW in 2 mg alum into only the left hind footpad or that of 25 μg RW in 2 mg alum into both the left hind footpad and the tail base induced significant infiltration of eosinophils into the conjunctiva of GKO mice, compared to WT mice. Conclusions: These results show that the immunization protocol employed has a marked effect on the severity of eosinophilic infiltration. These observations indicate that in interpreting experimental results in the study of EC, the immunization protocol employed must be considered.

Chlorpyrifos, an organophosphate pesticide, was evaluated for potential teratogenicity and developmental toxicity in mice. Pregnant females were given a single intraperitoneal injection (40 or 80 mg/kg) on day 10 of gestation and fetuses were evaluated on gestation day 17. At 80 mg/kg, chlorpyrifos treatment resulted in a significant reduction in numbers of live fetuses, and increase in resorptions, versus control litters. There was no indication of maternal toxicity. External and skeletal malformations were observed at 80 mg/kg, but not 40 mg/kg. Rates of fetuses with cleft palate increased significantly (p < 0.05) following 80 mg/kg chlorpyrifos (5.97%) versus control litters (0.97%). Similarly, the absence of thoracic vertebrae was increased and the number of caudal vertebrae was significantly decreased. It is suggested that chlorpyrifos is teratogenic and embryotoxic in mice at doses below those that cause significant maternal toxicity.

Endogenous interferon (IFN)-gamma negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN-gamma exerts its effects by binding to the IFN-gamma receptor (IFN-gammaR), the role that IFN-gammaR plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-gammaR and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-gammaR knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1-20 emulsified in Freund's complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN-gamma and tumor necrosis factor (TNF)-alpha] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN-gamma in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF-alpha were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-gammaR and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-gammaR and fyn may differ.

Under certain circumstances, fyn may serve to negatively regulate the differentiation of naive helper T (Th) cells into Th2 cells. This study aimed to investigate whether fyn negatively regulates the development of experimental immune-mediated blepharoconjunctivitis (EC), in which Th2 cells play an important role in C57BL/6 mice. C57BL/6 background wild-type (WT) or fyn knockout (fyn-/-) mice were subcutaneously immunized with ragweed (RW) adsorbed in aluminum hydroxide. Ten days later the mice were challenged with RW in eye drops, and 24 h after challenge, eyes, blood and spleens were harvested for histology, measurement of serum IgE, and proliferation or cytokine assays, respectively. RW-primed splenocytes from WT and fyn-/- mice were cultured in the presence of RW. Seventy-two hours later, either whole splenocytes or isolated CD4+T cells were transferred into syngeneic WT mice. Four days after the transfer, the recipient mice were challenged with RW and evaluated as described above. Infiltration of eosinophils into the conjunctiva induced by active immunization was significantly increased in fyn-/- mice relative to WT mice. Total serum IgE was also significantly higher in fyn-/- mice than in WT mice. In parallel, a higher level of IL-4 production from splenocytes was induced by concanavalin A stimulation in fyn-/- mice than in WT mice. In contrast to active immunization, transfer of whole splenocytes or separated CD4+T cells derived from WT or fyn-/- mice induced similar levels of eosinophilic infiltration in WT mice. Fyn regulates infiltration of eosinophils into the conjunctiva through downregulation of Th2 responses. This negative regulation is exerted only during the induction phase of EC.

The fatty acid composition, total phenol content and tocopherol content of olive oils from Shodo Island of Japan were analyzed for different varieties, maturation stages and extraction methods. Radical-scavenging activity of olive oils was also determined spectrophotometrically by measuring the disappearance of 1,1-diphenyl-2-picrylhydrazyl radical at 517nm. The differences in fatty acids composition, total phenol and tocopherol content in virgin olive oils were not great among the varieties. The content of oleic acid was in range of 66-78%. Though virgin olive oils contained more phenolic compounds than hexane-extracted olive oils, tocophenol content was not very different. In hexane-extracted olive oils, the total phenol content decreased during olive maturation. The tocopherol content of three types of olive oils also decreased during maturation. Still, large amounts of phenolic compounds remained in the olive residue after hexane extraction. The contribution of tocopherols to radical-scavenging activity was 39-61% in virgin olive oil, which suggests that both tocopherols and phenolic compounds contribute to radical-scavenging activity.

To evaluate fish as free radical scavengers in the human diet, we examined the radical-scavenging activity of some marine fish tissue commonly consumed in Japan, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH)-HPLC and 2'-deoxyguanosine oxidation method. All fishes showed high DPPH (259-2225 μmol Trolox eq./100g) and oxygen (1059-8018 μmol Trolox eq./100g) radical-scavenging activity. Ascorbic acid and tocopherol content were determined as free radical-scavenging compounds and ranged from 0.6-7.8 and 0.5-16.6 mg/100g fresh weight, respectively. Fish liver and skin showed higher contents of these antioxidative compounds than other parts, although their contribution to DPPH radical scavenging activity was negligible. Unlike vegetables some other compounds seem to be responsible for the high scavenging activity of fish.

Acute exposure of mice to potassium bromate (KBrO3), which is a major disinfection by-product of ozonation and/or chlorination of bromide-containing raw waters, causes serious kidney failure and neuropathological disorders. We observed significant elevations of serum uric acid levels and xanthine oxidase activity by KBrO3 administration (1.2 mmol/kg) with elevating relative kidney weight, serum creatinine levels and renal oxidative stress. Therefore, allopurinol was administered to KBrO3-treated mice to examine if the elevation of blood uric acid levels causes acute kidney damage and renal oxidative stress. These KBrO3-induced elevations were significantly prevented by intraperitoneal administration of allopurinol (10 or 50 mg/kg) and significant correlation between kidney damage and uric acid levels was observed. Reduction of catalase activity in the kidney of KBrO3-treated mice, which results in the accumulation of hydrogen peroxide, was also restored by allopurinol. There were significant correlations between catalase activity and uric acid levels or kidney damage. Furthermore, in in vitro experiment, catalase activity was reduced in the presence of physiological concentration of uric acid (approximately 0.3 mM or more). These results suggest that the reduction of catalase activity by the elevation of blood uric acid levels is a major cause of KBrO3-induced acute kidney damage. Allopurinol also suppressed KBrO3-induced increases of renal thiobarbituric acid reactive substances levels and renal protein carbonyl levels of mice. Furthermore, significant correlation between oxidative stress and blood uric acid levels was observed. Therefore, KBrO3 seems to cause hyperuricemic status which in turn brings about acute kidney damage and oxidative stress with reducing catalase activity.

Rutin, luteolin, luteolin-7-glucoside, and chlorogenic acid gradually decomposed during heating at 100°C. Even though they rapidly decomposed at 180°C, some decomposition products still had radical-scavenging activity. When rutin was heated in the presence of chlorogenic acid, decomposition of rutin was almost totally inhibited at 100°C, but was reduced at 180°C. These results suggest that the radical-scavenging activity is more stable than the content of original polyphenolic compounds in foods during cooking and processing.

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and polyphenol content of 25 dried fruits were evaluated and compared with fresh fruits. The contribution of ascorbic acid to this activity was also determined. All dried fruits contained DPPH radical-scavenging activity with hawthorn, apricot and blueberry having the highest. Other dried fruits containing rind also had high activity. The DPPH radical-scavenging activity and polyphenol content of dried fruits were highly correlated.

The effects of ascorbic acid and α-tocopherol on the antioxidant activity of 15 phenolic compounds were compared with 2 in vitro assays. Combination of ascorbic acid or α-tocopherol plus polyphenolic compounds resulted in an additive effect as shown with DPPH–HPLC method. With the liposome oxidation method, combination of quercetin or catechins plus α-tocopherol showed synergistic effects.

We employed a series of inhibitors of intracellular cascade to disclose the precise molecular mechanisms by which basic fibroblast growth factor (bFGF) promotes viability of PC12 cells and compared with nerve growth factor (NGF) and epidermal growth factor (EGF). The MEK 1 and 2 inhibitors, U0126 and PD98059, significantly suppressed cell viability mediated by bFGF in a dose-dependent manner, and to a greater extent compared with EGF and NGF. The degree of MEK dependency for growth factor-mediated cell viability was estimated to be in the order of bFGF, EGF, and NGF. Rapamycin strongly inhibited the effect of NGF on cell viability, compared with bFGF and EGF. The mechanisms of action of NGF-mediated cell viability may depend largely on p70 S6 kinase-related signal transduction pathways comparing to bFGF and EGF. The present findings suggest that different signal transduction systems may be involved in the molecular mechanisms by which bFGF, NGF, and EGF mediate cell viability.

The influence of polyphenol oxidase and ascorbate oxidase on radical-scavenging activity and contents of total phenol, chlorogenic acid, and ascorbic acid in vegetables during the cooking process were investigated. In the case of burdock and lettuce, which have a high activity of polyphenol oxidase, the radical-scavenging activity and the content of total phenol and chlorogenic acid decreased drastically within 1 min. In the case of broccoli, however, only a small decrease of radical-scavenging activity was observed, and total phenol and chlorogenic acid decreased almost not at all. The decrease of the activity in broccoli depended on the oxidation of ascorbic acid by ascorbate oxidase. None of these compounds decreased after the enzymes had been inactivated by heating.

The relationship of radical-scavenging activity and formation of brightly colored pigments in the early stage of the Maillard reaction was investigated. The Maillard reaction products of xylose with glycine, histidine, and arginine formed blue, yellow, and red color pigments, respectively, in the early stage. Although radical-scavenging activity was found in the early stages of the Maillard reaction, the scavenging activity appeared before the formation of the pigments. The radical-scavenging activity in the early stage of the Maillard reaction was derived from uncolored reaction products smaller than the brightly colored pigments. La relation entre activite antioxydante et formation de pigments colores dans les etapes precoces de la reaction de Maillard est etudiee. les produits formes a partir du xylose et de la glycine sont bleus, ceux formes en presence de l'histidine sont jaunes et ceux formes en presence d'arginine sont rouges. L'activite antioxydante apparait avant la formation des pigments et provient de produits de petite taille (plus petits que les pigments)

We reported large thyroid tumor in a 75-year old female who presented with cervical tumor. The patient first noticed the mass 30 years previously, however the mass had not enlarged. Then the tumor rapidly enlarged within one week with pain. On the initial examination in our hospital, the tumor was over 10 cm, reaching the submandibular gland. The tumor was diagnosed as thyroid malignant tumor by fine needle aspiration biopsy (FNA). The histological diagnosis suggested findings anaplastic carcinoma based on the present history, FNA findings and inflammatory response of blood examination. However, the tumor did not grow after administration and there was no accumulation on Ga scintigram. So we performed hemithyroidectomy alone after one week of administration, and the tumor was diagnosed as follicular adenoma with hematoma by intraoperative frozen section. We suggested that rapid enlargement of the tumor and inflammatory response was due to intratumoral hemorrhage, and misdiagnosis of FNA was due to a complicat

In developing embryos or in vitro differentiation cultures using pluripotent stem cells (PSCs), such as embryonic stem cells and induced pluripotent stem cells, fetal liver kinase 1 (Flk1)-expressing mesodermal cells are thought to be a heterogeneous population that includes hematopoietic progenitors, endothelial progenitors, and cardiac progenitors. However, information on cell surface markers for separating these progenitors in Flk1+ cells is currently limited. In the present study, we show that distinct types of progenitor cells in Flk1+ cells could be separated according to the expression of coxsackievirus and adenovirus receptor (CAR, also known as CXADR), a tight junction component molecule. We found that mouse and human PSC- and mouse embryo-derived Flk1+ cells could be subdivided into Flk1+CAR+ cells and Flk1+CAR− cells. The progenitor cells with cardiac potential were almost entirely restricted to Flk1+CAR+ cells, and Flk1+CAR− cells efficiently differentiated into hematopoietic cells. Endothelial differentiation potential was observed in both populations. Furthermore, from the expression of CAR, Flk1, and platelet-derived growth factor receptor-α (PDGFRα), Flk1+ cells could be separated into three populations (Flk1+PDGFRα−CAR− cells, Flk1+PDGFRα−CAR+ cells, and Flk1+PDGFRα+CAR+ cells). Flk1+PDGFRα+ cells and Flk1+PDGFRα− cells have been reported as cardiac and hematopoietic progenitor cells, respectively. We identified a novel population (Flk1+PDGFRα−CAR+ cells) with the potential to differentiate into not only hematopoietic cells and endothelial cells but also cardiomyocytes. Our findings indicate that CAR would be a novel and prominent marker for separating PSC- and embryo-derived Flk1+ mesodermal cells with distinct differentiation potentials. Significance Flk1-expressing (Flk+) mesodermal cells are assumed to be a heterogeneous population that includes hematopoietic progenitors and cardiac progenitors. However, information on cell surface markers for separating the Flk1+ cell subsets is currently limited. This study shows that hematopoietic lineage cells and cardiac lineage cells of pluripotent stem cell- and mouse embryo-derived Flk1+ cells could be separated based on the expression of coxsackievirus and adenovirus receptor (CAR), a tight junction component molecule. The results indicate that CAR is a novel and prominent cell surface marker for separating the Flk1+ cell subsets, and these differentiation methods based on the expression levels of CAR are expected to be instrumental in basic sciences and clinical studies.

International audience; Novel polyacetylenes bearing an optically active or racemic [6]helicene unit as the pendant groups directly bonded to the main-chain (poly-1s) were prepared by the polymerization of the corresponding acetylenes (1-rac, 1-P, and 1-M) with a rhodium catalyst. The optically active polyacetylenes (poly-1-P and poly-1-M) formed a preferred-handed helical conformation biased by the optically active helicene pendants, resulting in the induced circular dichroism (ICD) in their π-conjugated polymer backbone regions. The optically active helical polymers, when employed as an enantioselective adsorbent, showed a high chiral recognition ability towards racemates, such as the monomeric [6]helicene and 1,1’-binaphthyl analogues, and enantioselectively adsorbed one of the enantiomers.

The radical-scavenging activity of vegetables was measured using the 1,1-diphenyl-2-picrylhydrazyl-HPLC method, and the effect of cooking on this activity was determined. The content of ascorbic acid having radical-scavenging activity was highest in burdock among the 18 fresh vegetables tested. In some vegetables, the activity increased in spite of the loss of ascorbic acid content after boiling, while in other vegetables, the activity had decreased after boiling. This decrease may be due to release of the activity from cooked tissue into the cooking water during boiling. Both activity and ascorbic acid content of the vegetables cooked in a microwave were generally higher than in those cooked by boiling.

The role of high molecular weight compounds in the radical-scavenging activity of horse mackerel muscle was evaluated. After denaturation by boiling or by adding TCA solution, 30% of the total activity remained in the water extract. Fractions containing high molecular weight compounds showed both 1,1-diphenyl-2-picrylhydrazyl and peroxy radical-scavenging activity. These results indicate that water-soluble protein in fish muscle is responsible for the radical-scavenging activity.

肺切除術を受け合併症のなかった88名について術後早期の肺機能と運動耐容能を検討した.術後の評価は,平地歩行が普通にできるHugh-JonesのGrade 2になった時点とし,術後平均日数は17日であった.術後の肺機能は,肺活量は減少したが分時換気量が維持され,1回換気量の減少に対して呼吸数の増加による代償が認められることが示された.しかし,運動耐容能については,嫌気的代謝閾値での心拍数は術前後で差がなく,術後の酸素消費量と運動負荷量が低下した.この運動耐容能の低下は,文献的考察も加えたところ,換気当量および心拍出量の低下の影響が大きいと考えられた.

有機質肥料および化成肥料を用いて栽培されたキャベツとハクサイを試料とし,栽培条件の違いがラジカル捕捉活性,アスコルビン酸含量および生育状況に及ぼす影響について調べた.生育状況に関して,キャベッでは有機質肥料区より化成肥料区の方が全重が1.3～1.5倍重く,球高や球径もわずかに大きかった.一方,ハクサイでは有機質肥料栽培区の方が全重が重かった.ラジカル捕捉活性およびアスコルビン酸含量に対する肥料の影響を調べたところ,ラジカル捕捉活性はキャベツ,ハクサイともに有機質肥料区の方が化成肥料区より高い傾向を示したが有意差は認められなかった.アスコルビン酸含量についても栽培肥料の違いによる差異はみられなかった.また堆肥を併用した場合も,ラジカル捕捉活性やアスコルビン酸含量への影響は認められなかった.

The purpose of the present study was to examine whether cAMP-dependent protein kinase (PKA) was implicated in the process of long-term potentiation (LTP) in the hippocampal afferent fibre system to the prefrontal cortex in vivo. Using a biochemical approach, we measured PKA activity at different times after induction of LTP. We show that there is an NMDA receptor-dependent increase in PKA activity in the prefrontal cortex, only at five minutes after LTP induction. These data demonstrate a role of PKA in the induction and not the expression of cortical LTP and suggest that if PKA is involved in the late phase of LTP, it does not appear to be a persistent activation.

Embryonic stem (ES) cell- and induced pluripotent stem (iPS) cell-derived hematopoietic stem/progenitor cells (HSPCs) are considered as an unlimited source for HSPC transplantation. However, production of immature hematopoietic cells, especially HSPCs, from ES and iPS cells has been challenging. The adaptor protein Lnk has been shown to negatively regulate HSPC function via the inhibition of thrombopoietin (TPO) and stem cell factor signaling, and Lnk-deficient HSPCs show an enhanced self-renewal and repopulation capacity. In this study, we examined the role of Lnk on the hematopoietic differentiation from mouse ES and iPS cells by the inhibition of Lnk using a dominant-negative mutant of the Lnk (DN-Lnk) gene. We generated mouse ES and iPS cells stably expressing a DN-Lnk, and found that enforced expression of a DN-Lnk in ES and iPS cells led to an enhanced generation of Flk-1-positive mesodermal cells, thereby could increase in the expression of hematopoietic transcription factors, including Scl and Runx1. We also showed that the number of both total hematopoietic cells and immature hematopoietic cells with colony-forming potential in DN-Lnk-expressing cells was significantly increased in comparison with that in control cells. Furthermore, Lnk inhibition by the overexpression of the DN-Lnk gene augmented the TPO-induced phosphorylation of Erk1/2 and Akt, indicating the enhanced sensitivity to TPO. Adenovirus vector-mediated transient DN-Lnk gene expression in ES and iPS cells could also increase the hematopoietic cell production. Our data clearly showed that the inhibition of Lnk in ES and iPS cells could result in the efficient generation and expansion of hematopoietic cells.

Optically active, amphiphilic poly(meta-phenylene ethynylene)s (PPEa) bearing l- or d-alanine-derived oligo(ethylene glycol) side chains connected to the backbone via amide linkages were prepared by microwave-assisted polycondensation. PPEa’s exhibited an intense Cotton effect in the π-conjugated main-chain chromophore regions in various polar and nonpolar organic solvents due to a predominantly one-handed helical conformation stabilized by an intramolecular hydrogen-bonding network between the amide groups of the pendants. The stable helical structure was retained in the bulk and led to supramolecular column formation from stacked helices in oriented polymer films as evidenced by X-ray diffraction. Atomic force microscopy was used to directly visualize the helical structures of the polymers in two-dimensional crystalline layers with molecular resolution, and, for the first time, their absolute helical senses could unambiguously be determined.

Dysregulation of the G(1)/S transition in the cell cycle contributes to tumor development. The oncogenic transcription factors c-Jun and c-Myc are indispensable regulators at this transition, and their aberrant expression is associated with many malignancies. Degradation of c-Jun/c-Myc is a critical process for the G(1)/S transition, which is initiated upon phosphorylation by glycogen synthase kinase 3 β (GSK3β). However, a specific kinase or kinases responsible for priming phosphorylation events that precede this GSK3β modification has not been definitively identified. Here, we found that the dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 functions as a priming kinase of c-Jun and c-Myc. Knockdown of DYRK2 in human cancer cells shortened the G(1) phase and accelerated cell proliferation due to escape of c-Jun and c-Myc from ubiquitination-mediated degradation. In concert with these results, silencing DYRK2 increased cell proliferation in human cancer cells, and this promotion was completely impeded by codeprivation of c-Jun or c-Myc in vivo. We also found marked attenuation of DYRK2 expression in multiple human tumor samples. Downregulation of DYRK2 correlated with high levels of unphosphorylated c-Jun and c-Myc and, importantly, with invasiveness of human breast cancers. These results reveal that DYRK2 regulates tumor progression through modulation of c-Jun and c-Myc.

Various in vitro assays of active oxygen-scavenging activity in foods were compared by evaluating the activity of 13 food phenolic compounds and Trolox. 1,1-Diphenyl-2-picrylhydrazyl (DPPH)-HPLC and liposome oxidation methods were sensitive for these compounds, while deoxyribose oxidation and Randox kit methods were less sensitive. The deoxyguanosine oxidation method could not determine the activity since food polyphenols were pro-oxidative in this assay. Among these assays, the DPPH-HPLC method was shown to be the best since the liposome oxidation method is affected more by the structure of phenolic compounds than the DPPH-HPLC method. Dans cette etude, 5 dosages in vitro (DPPH-HPLC, desoxyribose oxydation, desoxyguanosine oxydation, liposome oxydation et kit Randox) sont compares en evaluant l'activite de captation d'oxygene actif de 10 polyphenols alimentaires et 3 acides monophenols. Le Trolox est aussi utilise car ce compose est souvent utilise comme antioxydant. La sensibilite de ces methodes est etudiee.

Transduction with replication-incompetent recombinant adenovirus (Ad) vectors results in a rapid activation of innate immune responses, such as inflammatory cytokine production and subsequent tissue damage. The precise mechanisms of the innate immune responses induced by Ad vectors remain to be clarified. Possible components of Ad vectors that activate innate immune responses are the capsid protein, the viral genome (DNA), and viral transcripts. In the present study, we demonstrate that virus-associated RNAs (VA-RNAs), which are small RNAs transcribed by RNA polymerase III, induce the production of type I IFN (IFN-α and IFN-β), but they do not induce the production of inflammatory cytokines (IL-6 and IL-12), in mouse embryonic fibroblasts (MEFs) and granulocyte-macrophage colony-stimulating factor–generated bone marrow-derived dendritic cells (GM-DCs). We also show that IFN-β promoter stimulator-1 is involved in VA-RNA–dependent IFN-β production in MEFs and is partially involved in type I IFN production in GM-DCs. This study provides important insight into the mechanisms of Ad vector-triggered innate immune responses, which may lead to more advanced and rational Ad vector designs for gene therapies and vaccine applications.

The purpose of this study was to determine the causes of visual impairment in Mie prefecture during a five-year period.The study was conducted between April 2004 and March 2009 in Mie Prefecture. 1,322 visually impaired people as defined by the Act on Welfare of Physically Disabled Persons were enrolled. We reviewed age, sex, causes of visual impairment, degree of disability and medical conditions according to their physical disability certificate.The four major causes of visual impairment were glaucoma (20.3%), diabetic retinopathy (18.9%), retinitis pigmentosa (12.2%), and macular degeneration (9.1%), followed by chorioretinal degeneration, stroke or brain tumor, optic atrophy, and cataract. The average ages of the four major causes were glaucoma (77.1 years), diabetic retinopathy (65.1 years), retinitis pigmentosa (62.5 years) and macular degeneration (77.7 years).The most common cause of visual impairment in Mie prefecture was glaucoma. The four major causes in Mie prefecture were the same as the results of the nation-wide investigation reported in 2006.

We have prepared ultra thin TiN films using an advanced ion-plating (AIP) apparatus developed by ShinMaywa Industries. In AIP, RF and/or DC bias voltages are applied to the substrate holder and then a capacitor is formed between the substrate holder and the chamber. Eventually, it becomes easy to sustain a stable plasma at Ar pressures at as low as 10 -3 Pa. We clarified the characteristics of the AIP method through the deposition of ultra thin TiN films and examined new application areas for TiN thin films. TiN thin films (thickness: 5 and 25 nni) were prepared by AIP and also by DC sputtering without substrate heating. Then we measured the resistivity and observed the nanometer scale surface morphology of the thin films. Although the initial resistivities of both films were close (200 x 10 -8 Ω.m), the resistivity of the sputtered film ruse to twice that of the AIP film 50 hours later. Atomic force microscopy measurements showed flat, fine grains spread uniformly over the surface in the AIP film, while many isolated crystal islands were formed in the sputtered film. Our findings indicated that the AIP films have a potential use as wiring material. We thought that the AIP deposition enhances surface migration more than sputtering does as a result of efficient excitation of impinging TiN clusters in the Ar plasma region.

Nuclear factor-κB (NF-κB) is tightly modulated by IκB kinases and IκBα in the cytoplasm. On stimulation, NF-κB translocates into the nucleus to initiate transcription; however, regulation of its transcriptional activity remains obscure. Here, we show that protein kinase C (PKC) δ controls the main subunit of NF-κB, RelA/p65. On exposure to tumor necrosis factor-α (TNF-α), the expression of RelA/p65 target genes such as IκBα, RelB, and p100/p52 is up-regulated in a PKCδ-dependent manner. The results also show that PKCδ is targeted to the nucleus and forms a complex with RelA/p65 following TNF-α exposure. Importantly, kinase activity of PKCδ is required for RelA/p65 transactivation. In concert with these results, PKCδ activates RelA/p65 for its occupancy to target-gene promoters, including IκBα and p100/p52. Moreover, functional analyses show that inhibition of PKCδ is associated with substantial attenuation of NF-κB activity in response to TNF-α. These findings provide evidence that PKCδ orchestrates RelA/p65 transactivation, a requisite for NF-κB signaling pathway in the nucleus. [Cancer Res 2009;69(14):5927–35]

The neuronal calcium binding protein calretinin was studied for possible effects on brain protein phosphorylation. Calretinin (100 nM) inhibited the appearance of a calcium stimulated 39 kDa phosphoprotein within a synaptic membrane fraction following sucrose density centrifugation. Calmodulin or a specific protein kinase C inhibitor had no effect on either the phosphorylation of the 39 kDa protein or the inhibition produced by calretinin. At the same concentration, calretinin produced a slight increase in the phosphorylation of several other synaptic membrane proteins which appeared additive with the stimulation produced by either calmodulin or phosphatidylserine in the presence of calcium.

Interleukin (IL)-6 has an important role in inflammatory diseases. Lysosomal enzymes cathepsins are widely expressed as cysteine proteases regulating inflammatory process. Caveolin-1 (Cav-1) is a scaffolding/regulatory membrane protein that interacts with signaling molecules. In this study, we investigated the role of Cav-1 on (1) the productivity, and (2) the enzymatic activity of cathepsin B and L in human gingival fibroblasts (HGFs) treated with IL-6 in the presence of soluble form of IL-6 receptor (sIL-6R). At first, we established the siRNA-mediated Cav-1 down-regulating in vitro systems by transient transfection of Cav-1 siRNA. The siRNA-mediated Cav-1 down-regulated cells were treated with IL-6/sIL-6R for indicated times. Then, cell lysates were collected, and examined the IL-6-induced signaling pathway, cathepsin B and L production, and measurement of cathepsins activity. To investigate the cathepsin L activity, cathepsin-(B + L) activity was measured after pretreatment with CA-074Me, a specific inhibitor for cathepsin B. We found that IL-6/sIL-6R enhanced significantly both production and activity of cathepsin B and L in HGFs. Interestingly, IL-6-mediated phosphorylation of both p44/42 MAPK and JNK was dramatically suppressed in Cav-1 down-regulated HGFs treated with IL-6/sIL-6R. In addition, both production and activity of cathepsin B and L were also significantly suppressed. Importantly, we demonstrated that JNK inhibition, but not p44/42 MAPK inhibition, significantly diminished IL-6/sIL-6R-induced cathepsin B and L production. Taken together, we concluded that IL-6/sIL-6R enhances cathepsin B and L production via IL-6/sIL-6R-mediated Cav-1-JNK-AP-1 pathway in HGFs. Our findings indicate that Cav-1 might be a therapeutic target for IL-6-mediated tissue degradation in periodontitis.

Adenovirus (Ad) vectors are among the most commonly used viral vectors in gene therapy clinical trials. However, the application of Ad vectors has been limited to local injection in many cases, because the systemic administration of Ad vectors triggers innate immune responses such as inflammatory cytokine production and tissue damage. To overcome this limitation, it will be necessary to develop safer Ad vectors less likely to induce the innate immune response. In the present study, we demonstrated that a suppressor of cytokine signaling-1 (SOCS1)-expressing Ad vector, Ad-SOCS1, reduces the innate immune response induced by Ad vectors. RAW264.7-SOCS1, a macrophage-like cell line that stably expresses SOCS1, was shown to produce lower levels of inflammatory cytokines after the transduction of Ad vectors. The systemic administration of Ad-SOCS1 into mice elicited the reduced production of inflammatory cytokines, as compared with that elicited by control Ad vectors, i.e., luciferase-expressing Ad vector, Ad-L2. Furthermore, the coadministration of Ad-L2 with Ad-SOCS1 attenuated inflammatory cytokine production and liver toxicity as compared with injection with Ad-L2 alone, and this was achieved without the suppression of luciferase production in various organs. The JAK/STAT pathway was involved in Ad vector-mediated cytokine production, which was impaired by the overexpression of SOCS1. These findings indicate that Ad-SOCS1 could be useful for reducing Ad vector-mediated innate immunity.

The aldehydes of yuzu peel oil were isolated and analyzed by GC, GC-MS, and NMR. Twenty-two aldehydes in yuzu peel oil of yellow and green maturity were identified. Three of them, 6-methyloctanal, 8-methylnonanal, and 8-methyldecanal, have not been previously reported to occur in nature. Further odor distinction beween green maturity and yellow maturity is also discussed

Post-translational modification and degradation of proteins by the ubiquitin-proteasome system are key regulatory events in cellular responses to various stimuli. The NF-kappaB signaling pathway is controlled by the ubiquitin-mediated proteolysis. Although mechanisms of ubiquitination in the NF-kappaB pathway have been extensively studied, deubiquitination-mediated regulation of the NF-kappaB signaling remains poorly understood. The present studies show that a deubiquitinating enzyme, USP11, specifically regulates IkappaB kinase alpha (IKKalpha) among the NF-kappaB signaling molecules. Knocking down USP11 attenuates expression of IKKalpha in the transcriptional, but not the post-translational, level. However, down-regulation of USP11 dramatically enhances NF-kappaB activity in response to tumor necrosis factor-alpha, indicating that IKKalpha does not require activation of NF-kappaB. Instead, knock down of USP11 or IKKalpha is associated with abrogation of p53 expression upon exposure to tumor necrosis factor-alpha. In concert with these results, silencing of USP11 is associated with transcriptional attenuation of the p53-responsive genes, such as p21 or Bax. Importantly, the ectopic expression of IKKalpha into cells silenced for USP11 restores p53 expression, demonstrating that USP11 functions as an upstream regulator of an IKKalpha-p53 signaling pathway.

A replication-incompetent adenoviral (Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 (MyD88)/Toll-like receptor (TLR)-9-dependent and/or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin (IL)-6 and IL-12 was significantly decreased in MyD88- or TLR9-deficient dendritic cells (DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88- or TLR9-deficient granulocyte-macrophage colony-stimulating factor (GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88- or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/TLR9-dependent and -independent mechanisms.

Protein kinase C delta (PKCdelta) functions as a redox-sensitive kinase in various cell types. Upon exposure to reactive oxygen species (ROS), it is activated by tyrosine phosphorylation, nuclear translocation and caspase-3-mediated cleavage. Activated PKCdelta is associated with cell cycle arrest or apoptosis, although its precise mechanism of action is unclear. Previous studies have demonstrated that the transcription factor, nuclear factor kappaB (NF-kappaB), functions as a redox-sensitive factor. ROS induce NF-kappaB signaling pathways including upstream IkappaB kinases (IKKs), although the mechanisms of ROS-induced activation of IKKs are unknown. Here we show that both PKCdelta and IKKalpha, but not IKKbeta, translocate to the nucleus in response to oxidative stress. The results also demonstrate that PKCdelta interacts with and activates IKKalpha. Importantly, our data suggest that, upon exposure to oxidative stress, PKCdelta-mediated IKKalpha activation does not contribute to NF-kappaB activation; instead, nuclear IKKalpha regulates the transcription activity of the p53 tumor suppressor by phosphorylation at Ser20. These findings collectively support a novel mechanism in which the PKCdelta-->IKKalpha signaling pathway contributes to ROS-induced activation of the p53 tumor suppressor.

We have recently identified lipopolysaccharide tumor-induced tumor necrosis factor alpha factor (LITAF) as a novel transcription factor controlling necrosis factor (TNF)-alpha expression in the human monocytic cell line, THP-1. To characterize the human (h) LITAF promoter, we isolated a 1.2-kb DNA fragment and followed this by a screening of human genomic DNA with a hLITAF cDNA probe. A 34-bp sequence domain located from nucleotides -74 to -43 in the hLITAF promoter exhibited the highest basal reporter gene activity; however, the activity was not elevated by lipopolysaccharide (LPS) stimulation. The sequence domain included a consensus sequence for hepatocyte nuclear factor (HNF)-3alpha, regulating the transcription of many kinds of genes. Interestingly, the DNA sequence position between -542 and -538 in the hLITAF promoter contained the CTCCC motif, which has been reported to act as a specific binding site for hLITAF protein. Electrophoretic mobility shift assays demonstrated that LPS induced the binding of THP-1 nuclear factors to a 22 bp probe containing the CTCCC motif. In addition, hLITAF mRNA and nuclear hLITAF protein increased significantly in the THP-1 cells immediately after LPS stimulation. These results suggest that the consensus sequence for HNF-3alpha, or a nuclear binding protein to the CTCCC motif, may play an important role in regulating LPS-dependent LITAF transcription.

Background: Eosinophils are important effector cells in severe allergic conjunctivitis such as vernal keratoconjunctivitis. Infiltration of eosinophils into the conjunctiva is mediated by type I and type IV allergic reactions. Cyclosporin A (CsA) eye drops are administered therapeutically for severe allergic conjunctivitis, but the mechanism by which CsA acts, that is, by inhibiting type I, type IV or both types of allergic reactions, is not known. We investigated whether CsA eye drops inhibit type I, type IV or both types of allergic reactions in the conjunctiva. Methods: Experimental immune-mediated blepharoconjunctivitis (EC) was induced in BALB/c mice by either active immunization or passive immunization by transfer of ragweed (RW)-primed splenocytes and RW-specific IgE, followed by RW challenge to the conjunctiva. These mice were treated in eye drops with vehicle, 0.1% CsA, 0.5% CsA or 0.1% betamethasone five times (1 and 2 h before RW challenge and 1, 2 and 3 h after RW challenge). Twenty-four hours after the challenge, the conjunctivas were harvested for histological analysis to evaluate eosinophilic infiltration. To evaluate effects of CsA eye drops on systemic immune responses, sera and spleens were collected from actively immunized mice at the time of sacrifice to examine serum IgE levels and cellular immune responses, respectively. Results: CsA eye drops significantly inhibited eosinophilic infiltration into the conjunctiva in actively immunized EC-developing mice compared with vehicle-treated mice. The CsA-induced inhibition was similar to inhibition induced by 0.1% betamethasone. Serum IgE levels and splenocyte responses in CsA-treated mice were equivalent to those in vehicle-treated mice. Betamethasone treatment inhibited eosinophilic infiltration into the conjunctiva induced by both splenocyte transfer and IgE transfer, while CsA treatment inhibited infiltration induced by splenocyte transfer. Conclusions: CsA eye drops inhibited eosinophilic infiltration into the conjunctiva without affecting systemic immune responses. CsA predominantly inhibits eosinophilic infiltration by interfering with the type IV allergic reaction in the conjunctiva.

When mice are treated with alpha-galactosylceramide (alpha-GalCer), NKT cells are activated and suppress the development of experimental airway inflammation. This suppressive effect is believed to be mediated by the upregulation of IFN-gamma. Here, we investigated whether alpha-GalCer treatment can also modulate the development of experimental allergic conjunctivitis (EC). EC was induced in wild-type and IFN-gamma-deficient Balb/c mice by active immunization with ragweed (RW) followed by challenge with RW in eye drops. The mice were intraperitoneally injected with alpha-GalCer or vehicle at the time of immunization or before RW challenge. Twenty-four hours after RW challenge, conjunctivas, spleens and sera were harvested for histological analysis, flow cytometric, proliferation and cytokine assays, and measurement of immunoglobulin levels, respectively. Treatment with alpha-GalCer at the time of the EC-priming immunization significantly increased Th2 responses and markedly upregulated the severity of the EC. However, treatment with alpha-GalCer just before the Ag challenge that triggers EC in primed animals significantly suppressed the disease. This was associated with an increased frequency of CD4(+)CD25(+) cells, which express Foxp3, in the spleen. alpha-GalCer treatment just prior to Ag challenge also suppressed the development of EC in IFN-gamma-deficient mice, and we found apoptosis and anergy are unlikely to play a major role in the mechanism by which pre-challenge alpha-GalCer treatment suppresses EC. These data suggest that NKT cells can play a downregulatory role in the development of EC and that alpha-GalCer may be useful for treating allergic conjunctivitis.