Mikinori Kuwabara

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes.

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum‐opsonized zymosan (sOZ) induced the activation of p38 mitogen‐activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3‐K) and sOZ‐induced O2 − production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3‐K). They caused significant attenuation of sOZ‐induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3‐K participated in both signaling pathways of NADPH oxidase activation (O2 − production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.

Radiation tolerance in the tardigrade Milnesium tardigradum

Purpose: Tardigrades are known to survive high doses of ionizing radiation. However, there have been no reports about radiation effects in tardigrades under culture conditions. In this study, we investigated tolerance of the tardigrade, Milnesium tardigradum, against gamma-rays and heavy ions by determining short-term or long-term survival, and reproductive ability after irradiation. Materials and methods: Hydrated and anhydrobiotic animals were exposed to gamma-rays (1000 – 7000 Gy) or heavy ions (1000 – 8000 Gy) to evaluate short-term survival at 2, 24 and 48 h post-irradiation. Long-term survival and reproduction were observed up to 31 days after irradiation with gamma-rays (1000 – 4000 Gy). Results: At 48 h after irradiation, median lethal doses were 5000 Gy (gamma-rays) and 6200 Gy (heavy ions) in hydrated animals, and 4400 Gy (gamma-rays) and 5200 Gy (heavy ions) in anhydrobiotic ones. Gamma-irradiation shortened average life span in a dose-dependent manner both in hydrated and anhydrobiotic groups. No irradiated animals laid eggs with one exception in which a hydrated animal irradiated with 2000 Gy of gamma-rays laid 3 eggs, and those eggs failed to hatch, whereas eggs produced by non-irradiated animals hatched successfully. Conclusion: M. tardigradum survives high doses of ionizing radiation in both hydrated and anhydrobiotic states, but irradiation with >1000 Gy makes them sterile.

Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells

OBJECTIVE In an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells. MATERIALS AND METHODS Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC). RESULTS In the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells. CONCLUSION This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.

The suppression of age-related accumulation of lipid peroxides in rat brain by administration of Rooibos tea (Aspalathus linearis)

The protective effects of Rooibos tea (RT), Aspalathus linearis, against damage to the central nervous system (CNS) accompanying aging were examined by both the thiobarbituric acid reaction (TBA) and magnetic resonance imaging (MRI) methods in brains of chronically RT-treated rats. Ad libitum administration of RT was begun with 3-month-old Wistar female rats and continued for 21 months. The contents of TBA reactive substances (TBARS) in the frontal cortex, occipital cortex, hippocampus and cerebellum in 24-month-old rats after administration with water were significantly higher than those in young rats (5 weeks old). However, no significant increase of TBARS was observed in RT-administered aged rats. When MR images of the brains of 24-month-old rats with and without RT as well as 5-week-old rats were taken, a decrease of the signal intensity was observed in the cerebral cortex, hippocampus and cerebellum in MR images of aged rats without RT, whereas little change of the signal intensity was observed in MR images of the same regions of 24-month-old rats treated with RT, whose images were similar to those of young rats. These observations suggested that (1) the age-related accumulation of lipid peroxides in the brain was closely related to the morphological changes observed by MRI, and (2) chronic RT-administration prevented age-related accumulation of lipid peroxides in several regions of rat brain.

Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells.

Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl-L-cysteine (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X(L) and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6h after X irradiation. Furthermore, the O(2)(-*) production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO-OOH was detected. This NADH/succinate-dependent O(2)(-*) production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O(2)(-*) production from mitochondria to trigger cytochrome c release in A549 cells.

Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion

BACKGROUND: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum‐free medium. However, the stromal cells were prepared using long‐term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum‐based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted.

Neuroprotective effect of α-phenyl-N-tert-butylnitrone in gerbil hippocampus is mediated by the mitogen-activated protein kinase pathway and heat shock proteins

α-Phenyl-N-tert-butylnitrone (PBN), a spin trap, is known as a protective agent against delayed-neuronal death after ischemia-reperfusion. To investigate this neuroprotective effect of PBN, we examined the effect of PBN on the mitogen-activated protein kinase (MAPK) signaling pathway and the expression of heat shock proteins (HSPs) in the gerbil hippocampus following transient (5 min) ischemia. Immunoblot analysis revealed that intraperitoneal (i.p.) injection of PBN (200 mg/kg) enhanced the activation of extracellular-response kinase (ERK) and suppressed the activation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (p38) at 6 h after ischemia. Elevated levels of HSP27 and HSP70 were seen at the same period. These data suggest that PBN protects against delayed-neuronal death not only by its inherent radical-trapping activity but also by regulating the MAPK pathway and up-regulating HSPs.

Stromal cell-dependent ex vivo expansion of human cord blood progenitors and augmentation of transplantable stem cell activity

In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications. Thrombopoietin (TPO) and flt3/flk2 ligand (FL) have been suggested to support the proliferation of primitive hematopoietic progenitors and the expansion of transplantable stem cells in culture. In this study, we examined the synergistic effects of the murine stromal cell line MS-5 and a combination of the two cytokines, TPO and FL, on the ex vivo expansion of human cord blood primitive progenitors and transplantable stem cells. A monolayer of MS-5 cells with TPO/FL synergistically supported a more than 600-fold expansion of human cord blood CD34+ cells and CD34+CD38− cells in 2 weeks of culture. Colony-forming unit in culture (CFU-C) and 5-week and 8-week cobblestone area-forming cells (CAFC) were also expanded approximately 300-, 4- and 13-fold, respectively. When MS-5 cells were physically separated from progenitors by a Transwell filter, the synergy was reduced to a quarter of the control, suggesting that direct cell–cell contact between MS-5 cells and progenitors is required for maximum expansion. The severe-combined immunodeficient (scid) mouse-reconstituting cell (SRC) assay demonstrated the slight augmentation of transplantable stem cell activity in culture. These results indicated that MS-5 cells provide a milieu that stimulates the proliferation of primitive progenitors including transplantable stem cells. Bone Marrow Transplantation (2000) 26, 837–844.

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.

Roles of protein kinase C delta in the accumulation of P53 and the induction of apoptosis in H2O2-treated bovine endothelial cells.

To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H 2 O 2 and investigated the roles of protein kinase C i (PKC i ) and Ca 2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H 2 O 2 caused the accumulation of p53, which was inhibited by rottlerin (a PKC i inhibitor) but not by BAPTA-AM (an intracellular Ca 2+ chelator). PKC i itself was activated through the phosphorylation at tyrosine residues. H 2 O 2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKC i contributes to the accumulation of p53 and that Ca 2+ plays a role in downstream signals of p53 leading to apoptosis in H 2 O 2 -treated BAEC.