Tat-PRAS40 prevent hippocampal HT-22 cell death and oxidative stress induced animal brain ischemic insults.

Proline rich Akt substrate (PRAS40) is a component of mammalian target of rapamycin complex 1 (mTORC1) and is known to play an important role against reactive oxygen species-induced cell death. However, the precise function of PRAS40 in ischemia remains unclear. Thus, we investigated whether Tat-PRAS40, a cell-permeable fusion protein, has a protective function against oxidative stress-induced hippocampal neuronal (HT-22) cell death in an animal model of ischemia. We showed that Tat-PRAS40 transduced into HT-22 cells, and significantly protected against cell death by reducing the levels of H2O2 and derived reactive species, and DNA fragmentation as well as via the regulation of Bcl-2, Bax, and caspase 3 expression levels in H2O2 treated cells. Also, we showed that transduced Tat-PARS40 protein markedly increased phosphorylated RRAS40 expression levels and 14-3-3σ complex via the Akt signaling pathway. In an animal ischemia model, Tat-PRAS40 effectively transduced into the hippocampus in animal brain and significantly protected against neuronal cell death in the CA1 region. We showed that Tat-PRAS40 protein effectively transduced into hippocampal neuronal cells and markedly protected against neuronal cell damage. Therefore, we suggest that Tat-PRAS40 protein may be used as a therapeutic protein for ischemia and oxidative stress-induced brain disorders.

Transduced PEP-1-FK506BP ameliorates atopic dermatitis in NC/Nga mice.

Immunophilin, FK506-binding protein 12 (FK506BP), is a receptor protein for the immunosuppressive drug FK506 by the FK506BP/FK506 complex. However, the precise function of FK506BP in inflammatory diseases remains unclear. Therefore, we examined the protective effects of FK506BP on atopic dermatitis (AD) in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced HaCaT cells and 2,4-dinitrofluorobenzene-induced AD-like dermatitis in Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice using a cell-permeable PEP-1-FK506BP. Transduced PEP-1-FK506BP significantly inhibited the expression of cytokines, as well as the activation of NF-κB and mitogen-activated protein kinase (MAPK) in TNF-α/IFN-γ-induced HaCaT cells. Furthermore, topical application of PEP-1-FK506BP to NC/Nga mice markedly inhibited AD-like dermatitis as determined by a histological examination and assessment of serum IgE levels, as well as cytokines and chemokines. These results indicate that PEP-1-FK506BP inhibits NF-κB and MAPK activation in cells and AD-like skin lesions by reducing the expression levels of cytokines and chemokines, thus suggesting that PEP-1-FK506BP may be a potential therapeutic agent for AD.

Anti-oxidative effects of Phellinus linteus and red ginseng extracts on oxidative stress-induced DNA damage.

Anti-oxidative effect of Phellinus linteus (P. linteus) and red ginseng extracts on DNA damage induced by reactive oxygen species (ROS) were investigated in this study. P. linteus (PLE) and red ginseng extracts (RGE) inhibited the breaking of E. coli ColE1 plasmid DNA strands as well as nuclear DNA of rat hepatocytes damaged by oxidative stress. In addition, a reaction mixture of PLE and RGE showed synergistic inhibitory effect against DNA damage. These results suggest that PLE and RGE have a cellular defensive effect against DNA damage induced by ROS.

Human brain pyridoxal-5'-phosphate phosphatase: production and characterization of monoclonal antibodies.

We cloned and expressed human pyridoxal-5\'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin B6, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin B6 abnormalities.

Copper chaperone for Cu,Zn-SOD supplement potentiates the Cu,Zn-SOD function of neuroprotective effects against ischemic neuronal damage in the gerbil hippocampus.

In the present study, we investigated the chronological alterations in SOD1 and its copper chaperone (chaperone for superoxide dismutase, CCS) immunoreactivities and their neuroprotective effects against neuronal damage in the gerbil hippocampus after 5 min of transient forebrain ischemia. SOD1 and CCS immunoreactivities were significantly increased in the stratum pyramidale of the CA1 region at 24 and 12 h after ischemic insult, respectively. At 24 h after ischemic insult, the SOD1 and CCS immunoreactivities were colocalized in the CA1 pyramidal cells of the stratum pyramidale. Thereafter, their immunoreactivities were significantly decreased in the CA1 region. To elucidate the effects of CCS or CCS/SOD1, we constructed the expression vectors PEP-1-SOD and PEP-1-CCS. In the CCS-treated group and the CCS/SOD1-treated group, 43.9 and 78.9% pyramidal cells, respectively, compared to the sham-operated group, were stained with cresyl violet 5 or 7 days after ischemic insult. The distribution pattern of active astrocytes and microglia in the PEP-CCS/SOD1-treated group 5 days after ischemic insult was similar to that of the sham-operated group. In addition, the SOD activity in the PEP-CCS- or PEP-CCS/SOD1-treated group was maintained by 10 days after ischemic insult. The SOD activity was higher in the PEP-CCS/SOD1-treated group vs the CCS-treated group. These results suggest that the enhanced expression of SOD1 and CCS may be related to compensatory mechanisms against ischemic damage and that cotreatment with CCS and SOD1 has a greater neuroprotective effect than treatment with CCS or SOD1 in isolation.