Takashi Okamoto

Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection.

AIDS and the bare lymphocyte syndrome (BLS) are severe combined immunodeficiencies. BLS results from mutations in genes that regulate the expression of class II major histocompatibility (MHC II) determinants. One of these is the class II transactivator (CIITA). HIV and its transcriptional transactivator (Tat) also block the expression of MHC II genes. By binding to the same surface in the cyclin T1, which together with CDK9 forms the positive transcription elongation factor b (P-TEFb) complex, Tat inhibits CIITA. CIITA can also activate transcription when tethered artificially to RNA. Moreover, a dominant-negative CDK9 protein inhibits the activity of MHC II promoters. Thus, CIITA is a novel cellular coactivator that binds to P-TEFb for the expression of its target genes.

The role of p38 mitogen‐activated protein kinase in IL‐6 and IL‐8 production from the TNF‐α‐ or IL‐1β‐stimulated rheumatoid synovial fibroblasts

We examined the role of p38 mitogen‐activated protein (MAP) kinase in the tumor necrosis factor α (TNF‐α)‐ or interleukin‐1β (IL‐1β)‐induced production of interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL‐6 and IL‐8 without affecting nuclear translocation of nuclear factor κB (NF‐κB) or IL‐6 and IL‐8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.

Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line

Exposure of the human skin to ultraviolet radiation (UVR) leads to depletion of cutaneous antioxidants, regulation of gene expression and ultimately to the development of skin diseases. Although exogenous supplementation of antioxidants prevents UVR-induced photooxidative damage, their effects on components of cell signalling pathways leading to gene expression has not been clearly established. In the present study, the effects of the antioxidants alpha-lipoic acid, N-acetyl-L-cysteine (NAC) and the flavonoid extract silymarin were investigated for their ability to modulate the activation of the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in HaCaT keratinocytes after exposure to a solar UV simulator. The activation of NF-kappaB and AP-1 showed a similar temporal pattern: activation was detected 2 h after UV exposure and maintained for up to 8 h. To determine the capacity of activated NF-kappaB to stimulate transcription, NF-kappaB-dependent gene expression was measured using a reporter gene assay. The effects of the antioxidants on NF-kappaB and AP-1 activation were evaluated 3 h after exposure. While a high concentration of NAC could achieve a complete inhibition, low concentrations of alpha-lipoic acid and silymarin were shown to significantly inhibit NF-kappaB activation. In contrast, AP-1 activation was only partially inhibited by NAC, and not at all by alpha-lipoic acid or silymarin. These results indicate that antioxidants such as alpha-lipoic acid and silymarin can efficiently modulate the cellular response to UVR through their selective action on NF-kappaB activation.

NF-κB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2

Nuclear factor κB (NF-κB) is a transcription factor that controls the expression of many cellular and viral genes. The p65 (RelA) subunit plays a critical role as a transcriptional activator and recent observations have highlighted its role in the control of apoptosis. Here we report that 53BP2, a protein previously identified by interaction with wild type p53 and Bcl-2, also binds to p65 in a yeast two-hybrid system. This specific interaction was confirmed by pull-down assay in vitro and by a mammalian two-hybrid assay in vivo. We observed that full-length 53BP2 fused to GFP had a punctate distribution in cytoplasm, predominantly in perinuclear region whereas the N-terminal 53BP2 localized in cytoplasm and C-terminal 53BP2 localized in the nucleus. Furthermore, we found that overexpression of GFP-53BP2 induced apoptosis in transiently transfected cells. Neither the N-terminal nor the C-terminal of 53BP2 fused to GFP induced cell death. Interestingly, co-transfection with a p65 expression plasmid significantly inhibited 53BP2-induced cell death. The previous findings that 53BP2 bound to p53 and Bcl-2 together with our present observations suggest that 53BP2 may play a central role in the regulation of apoptosis and cell growth.

A potential role of activated NF-κB in the pathogenesis of euthyroid sick syndrome

Euthyroid sick syndrome, characterized by low serum 3,5, 3-triiodothyronine (T(3)) with normal L-thyroxine levels, is associated with a wide variety of disorders including sepsis, malignancy, and AIDS. The degree of low T(3) in circulation has been shown to correlate with the severity of the underlying disorders and with the prognosis. Elevated TNF-alpha levels, which accompany severe illness, are associated with decreased activity of type I 5-deiodinase (5-DI) in liver, leading us to speculate that high levels of this factor contribute to euthyroid sick syndrome. Here we demonstrate that the activation of NF-kappa B by TNF-alpha interferes with thyroid-hormone action as demonstrated by impairment of T(3)-dependent induction of 5-DI gene expression in HepG2 cells. Inhibition of NF-kappa B action by a dominant-negative NF-kappa B reversed this effect and allowed T(3) induction of 5-DI. Furthermore, we show that an inhibitor of NF-kappa B activation, clarithromycin (CAM), can inhibit TNF-alpha-induced activation of NF-kappa B and restore T(3)-dependent induction of 5-DI mRNA and enzyme activity. These results suggest that NF-kappa B activation by TNF-alpha is involved in the pathogenesis of euthyroid sick syndrome and that CAM could help prevent a decrease in serum T(3) levels and thus ameliorate euthyroid sick syndrome.

Selective inhibition of NF-κB activation by the flavonoid hepatoprotector silymarin in HepG2: Evidence for different activating pathways

The bioflavonoid silymarin is found to potently suppress both nuclear factor kappa‐B (NF‐κB)‐DNA binding activity and its dependent gene expression induced by okadaic acid in the hepatoma cell line HepG2. Surprisingly, tumor necrosis factor‐α‐induced NF‐κB activation was not affected by silymarin, thus demonstrating a pathway‐dependent inhibition by silymarin. Many genes encoding the proteins of the hepatic acute phase response are under the control of the transcription factor NF‐κB, a key regulator in the inflammatory and immune reactions. Thus, the inhibitory effect of silymarin on NF‐κB activation could be involved in its hepatoprotective property.

Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB

The transcription factor NF-κB is a positive transcription factor for a number of genes and has been recognized as an anti-apoptotic regulator. However, the mechanism by which NF-κB blocks apoptosis is still controversial. Here, we demonstrate the evidence that NF-κB could attenuate the TNF-α-induced apoptosis without de novo protein synthesis using human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. The TNF-α-induced apoptosis was blocked by IL-1β, a potent inducer of NF-κB activation. This inhibitory effect of IL-1β was evident when cells were treated with protein synthesis inhibitors such as cycloheximide (CHX). Moreover, NF-κB decoy oligonucleotides could not block the anti-apoptotic effect of IL-1β at doses sufficient to block the NF-κB-dependent transcription induced by IL-1β. To confirm the role of NF-κB in blocking apoptosis, we generated stable cell lines expressing IκBΔN, a highly stable form of IκBα, a cytoplasmic inhibitor of NF-κB. In these stable transfectants, the anti-apoptotic effect of IL-1β was totally abolished, indicating that the anti-apoptotic action of IL-1β could be ascribed to the NF-κB action. These findings show that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB and support the possibility that NF-κB could exert its anti-apoptotic action through protein-protein interaction.

Conservation of the central proline-rich (PxxP) motifs of human immunodeficiency virus type 1 Nef protein during the disease progression in two hemophiliac patients

The nef gene is considered to play a crucial role in the development of acquired immunodeficiency syndrome (AIDS). In this study, we analyzed the sequence of nef quasispecies obtained from replication‐competent HIV‐1 isolates from two Japanese hemophiliac patients infected with HIV‐1. At least 10 nef clones were isolated at each time point and a total of 75 individual nef quasispecies were sequenced. We observed a gradual increase in genetic diversity of the nef gene over time. Among the various functional regions of Nef protein, myristoylation site and the central PXXP (SH3 ligand) motifs were well conserved. The scattered regions responsible for downregulation of CD4 and class I MHC were also conserved. These data suggest that these functions of Nef may be involved throughout the disease process.

Role of thioredoxin in the redox regulation of gene expression in inflammatory diseases

Oxygen is an essential molecule to generate energy for all aerobic life forms including humans. However, while oxygen is indispensable for such cells to obtain the essential chemical energy as a form of adenosine triphosphate (ATP), which is called respiration, incomplete reduction of oxygen generates highly active, as well as toxic, forms called radical oxygen intermediates (ROI). In addition to being produced as physiological side-products of electron transfer reactions using oxygen as an electron acceptor, ROI are produced in the cell by various environmental stimuli such as infection of microbes (viruses, bacteria, etc.), ionizing and UV irradiation, and pollutants (i.e. oxidants), which are collectively called “oxidative stress”. These ROI are highly reactive with biological macromolecules, producing lipid peroxides (which are often radicals), inactivating proteins and mutating DNA (by producing 8-hydroxy-deoxy-guanosine, 8-OH-dG, or breaking nucleic acid chains).

EP-1500: Development of tumor response observation system for dose-volume delivery guided particle therapy

Results: The measured a photoluminescence peak value of the Gd2O3:Eu was 611nm, which was identical with literature value. In case of the calculated value using GEANT4 montecarlo code, an intensity(counting) of the photoluminescence peak value was 2 times higher, but the peak value also was identical with measured the peak value and overall trend of the photoluminescence spectrum was correspond to the measured data. A result of the decay time showed that the measured value was 1.2 times higher than that of the calculated value despite the higher intensity, but the measured and calculated value was well matched in low intensity.