Kei-ichi Sugiyama

Aurora-B associated protein phosphatases as negative regulators of kinase activation

The human serine/threonine kinase Aurora-B is structurally related to the protein kinase Ipl1p from S cerevisiae and aurora from Drosophila melanogaster, which are key regulators of mitosis. The present study shows that human Aurora-B is activated by okadaic acid and forms complexes with the protein serine/threonine phosphatase type 1 (PP1) or PP2A, but not with PP5. These data identified Aurora-B associated protein phosphatases as negative regulators of kinase activation. We then used a series of substrates based on a histone H3 phosphorylation site (residues 5–15) to determine the substrate specificity of human Aurora-B. We found that this enzyme is an arginine-directed kinase that can phosphorylate histone H3 at serines 10 and 28 in vitro, suggesting that human Aurora-B is a mitotic histone H3 kinase.

Role of Toll‐Like Receptor 4 in Mediating Multiorgan Dysfunction in Mice With Acetaminophen Induced Acute Liver Failure

Strategies for the prevention of multiorgan dysfunction (MOD) in acetaminophen (APAP)‐induced acute liver failure (ALF) are an unmet need. Our study tested the hypothesis that sterile inflammation induced by APAP in a mouse model would activate toll‐like receptor 4 (TLR4) in the liver and extrahepatic organs and lead to the progression of ALF and MOD and that the administration of the novel TLR4 antagonist STM28 (a peptide formed of 17 amino‐acids) would prevent liver injury and associated MOD. ALF and, subsequently, MOD were induced in TLR4‐knockout (KO) mice (B6.B10ScN‐Tlr4 lpsdel /JthJ) and wild‐type (WT) mice (C57BL/6) with APAP (500 mg/kg). A second set of experiments was conducted to evaluate the effects of a pretreatment with a novel TLR4 antagonist, STM28, on APAP‐induced MOD in CD1 mice. Animals were sacrificed at the coma stage, and plasma, peripheral blood cells, liver, kidneys, and brain were collected. Biochemistry values and cytokines were measured. Liver and kidneys were studied histologically and were stained for TLR4 and activated Kupffer cells, and the expression of nuclear factor kappa B–p65 was quantified with western blotting. Brain water was measured in the frontal cortex. After APAP administration, TLR4‐KO (NFkBp65) mice were relatively protected from liver necrosis and end‐organ dysfunction and had significantly better survival than WT controls (P < 0.01). STM28 attenuated liver injury and necrosis, reduced creatinine levels, and delayed the time to a coma significantly. The increases in cytokines in the plasma and liver, including TLR4 expression and the activation of Kupffer cells, after APAP administration were reduced significantly in the STM28‐treated animals. The increased number of circulating myeloid cells was reduced significantly after STM28 treatment. In conclusion, these data provide evidence for an important role of the TLR4 antagonist in the prevention of the progression of APAP‐induced ALF and MOD. Liver Transpl 19:751–761, 2013..

A novel TLR4-binding peptide that inhibits LPS-induced activation of NF-κB and in vivo toxicity

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. It is a ligand for Toll-like receptor 4 (TLR4), which plays an essential role in innate immunity. Macrophages and dendritic cells exposed to LPS overproduce proinflammatory mediators, leading to septic shock. In this study, we screened for peptides that associate with TLR4 with a yeast two-hybrid screen using the human TLR4 extracellular domain as bait. A peptide (STM28) isolated from the screen inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in human and mouse macrophage cells and interacted with TLR4 in yeast and mammalian cells. STM28 showed no inhibitory effects against NF-kappaB activation induced by TLR1/2, TLR3 and TLR9 ligands in a mouse macrophage cell line, RAW 264. In addition, STM28 suppressed LPS-induced tumor necrosis factor-alpha production by differentiated THP-1 cells. Moreover, LPS-induced lethality in d-galactosamine-sensitized mice was significantly repressed by STM28 in a dose-dependent manner. These results demonstrate that STM28 selectivity inhibits TLR4-induced macrophage activation, and suggest that STM28 may have utility as a novel therapeutic agent for Gram-negative bacterial sepsis.

NF-κB activation via MyD88-dependent Toll-like receptor signaling is inhibited by trichothecene mycotoxin deoxynivalenol

Macrophages induce the innate immunity by recognizing pathogens through Toll-like receptors (TLRs), which sense pathogen-associated molecular patterns. Myeloid differentiation factor 88 (MyD88), which is an essential adaptor molecule for most TLRs, mediates the induction of inflammatory cytokines through nuclear factor κB (NF-κB). Trichothecene mycotoxin deoxynivalenol (DON) shows immunotoxic effects by interrupting inflammatory mediators produced by activated macrophages. The present study investigates the effect of DON on NF-κB in activated macrophages through MyD88-dependent pathways. DON inhibited NF-κB-dependent reporter activity induced by MyD88-dependent TLR agonists. In addition, lipopolysaccharide-induced phosphorylation of interleukin-1 receptor-associated kinase 1 and inhibitor κBα were attenuated by DON. Furthermore, DON downregulated the expression level of MyD88. These results suggest that DON inhibits NF-κB activation in macrophages stimulated with TLR ligands via MyD88-dependent TLR signals. Therefore exposure to DON may lead to the inhibition of MyD88-dependent pathway of TLR signaling.

Mutagenicity of ω-3 fatty acid peroxidation products in the Ames test

Polyunsaturated fatty acids (PUFA) represent one of the main building blocks of cellular membranes and their varying composition impacts lifespan as well as susceptibility to cancer and other degenerative diseases. Increased intake of ω-3 PUFA is taught to compensate for the abundance of ω-6 PUFA in modern human diet and prevent cardiocirculatory diseases. However, highly unsaturated PUFA of marine and seed origin easily oxidize to aldehydic products which form DNA adducts. With increased PUFA consumption it is prudent to re-evaluate ω-3 PUFA safety and the genotoxic hazards of their metabolites. We have used the standard Ames test to examine the mutagenicity of 2 hexenals derived from lipid peroxidation of the common ω-3 PUFA in human diet and tissues. Both 4-hydroxyhexenal and 2-hexenal derived from the ω-3 docosahexaenoic and α-linolenic acid, respectively, induced base substitutions in the TA104 and TA100 Ames strains in a dose dependent manner. Their mutagenicity was dependent on the Y-family DNA polymerase RI and they did not induce other types of mutations such as the -2 and -1 frameshifts in the TA98 and TA97 strains. Our results expand previous findings about the mutagenicity of related ω-3 peroxidation product 4-oxohexenal and raise alert that overuse of ω-3 rich oils may have adverse effect on genome stability.

Inhibitory effect of citrinin on lipopolisaccharide-induced nitric oxide production by mouse macrophage cells

The present study evaluated the immunotoxicity of citrinin (CIT), a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. Because nitric oxide (NO), a pro-inflammatory mediator, plays an important role in the protection from pathogens, we addressed the effect of CIT on NO production by a mouse macrophage-like cell line RAW264 activated with lipopolysaccharide (LPS). LPS-induced NO release from RAW264 cells was inhibited by CIT. Moreover, the transcription and expression of inducible NO synthase (iNOS) by LPS was suppressed by CIT. These results show that CIT suppressed the LPS-induced NO production and iNOS expression, which contribute to the host protection against invading pathogens. This suggests that CIT on LPS-induced NO release may exert adverse effects in macrophages, indicating immunotoxic effects of this toxin. .

Effect of a combination of deoxynivalenol and nivalenol on lipopolisaccharide-induced nitric oxide production by mouse macrophages

Deoxynivalenol (DON) and nivalenol (NIV) are trichothecene mycotoxins produced by Fusarium fungi as secondary metabolites. Both compounds have the immunotoxic effects that the productions of inflammatory mediators by activated macrophages is disturbed. Co-contamination with DON and NIV can occur; however, the effects of simultaneous contamination are not well known. The present study investigated the combined effects of DON and NIV on nitric oxide (NO) production by mouse macrophages stimulated with lipopolisaccharide (LPS). The inhibitory effect of DON and NIV on NO release from activated macrophages has already been reported as an appropriate indicator of immunotoxic effect of the both compounds. LPS-induced NO production in macrophages was inhibited by both of these toxins individually in a dose-dependent manner, and toxin mixtures at the same concentration inhibited NO production in the same manner. In addition, there were no unique inhibitory effects on LPS-induced NO production in macrophages in the presence of mixtures of various molar ratios. These results suggest that the combined effects of DON and NIV can be predicted based on addition of each compound alone.

Detection of epigenetic mutagens including anthracene-derived compounds using yeast FLO1 promoter GFP reporter gene assay

Recently, we have reported that the FLO1-mediated flocculation levels of yeast are affected by an epigenetic mutagen, alizarin. Alizarin promoted flocculation and reduced the bulk levels of histone H3 in yeast cells. Since alizarin has been known to possess carcinogenesis-promoting properties, it is important to estimate the effect of alizarin-related compounds on epigenome as measured by the flocculation of yeast. In this study, we examined the effects of two anthracene-derived compounds other than alizarin on the flocculation level of yeast. Purpurin significantly promoted the flocculation in a dose-dependent manner. While, quinizarin had a weaker promoting effect than purpurin. The strain treated with purprin showed FLO1 mRNA upregulation and reduced histone H3 expression similarly to alizarin. We also confirmed that the purprin-treated cells frequently exhibited abnormally shaped nuclei. Moreover, fluorescence intensities of green fluorescent protein (GFP) reporter under the FLO1 promoter control were dose-dependently increased by purprin and alizarin in the yeast. Taken together, these results suggest that the GFP reporter gene system utilising the FLO1 promoter is useful for the detection of epigenetic mutagens including anthracene-derived compounds.

Opposing roles of Y-family DNA polymerases in lipid peroxide mutagenesis at the hisG46 target in the Ames test

DNA polymerases play a key role in mutagenesis by performing translesion DNA synthesis (TLS). The Y-family of DNA polymerases comprises several evolutionarily conserved families, specializing in TLS of different DNA adducts. Exocyclic etheno and propano DNA adducts are among the most common endogenous DNA lesions induced by lipid peroxidation reactions triggered by oxidative stress. We have investigated the participation of two enterobacterial representatives of the PolIV and PolV branches of Y-family DNA polymerases in mutagenesis by two model lipid peroxidation derived genotoxins, glyoxal and crotonaldehyde. Mutagenesis by the ethano adduct (glyoxal-derived) and the propano adduct (crontonaldehyde-derived) at the GC target in the Ames test depended exclusively on PolV type DNA polymerases such as PolRI. In contrast, PolIV suppressed glyoxal and, even more, crotonaldehyde mutagenesis, as detected by enzyme overexpression and gene knockout approaches. We propose that DNA polymerase IV, which is the mammalian DNA polymerase κ ortholog, acts as a housekeeper protecting the genome from lipoxidative stress.

Characterization of Sarcocystis fayeri's actin-depolymerizing factor as a toxin that causes diarrhea

Raw horsemeat has the potential to induce food poisoning which often presents with diarrheal symptoms. A sample of horsemeat was found to be infected with Sarcocystis fayeri, and a 15‐kDa protein isolated from the cysts of S. fayeri was found to clearly show its diarrhea‐inducing activity. A nested polymerase chain reaction was used to clone the cDNA of the 15‐kDa protein. The deduced amino acid sequence showed homology to actin‐depolymerizing factor (ADF). A recombinant 15‐kDa protein depolymerized prepolymerized actins in a test tube. The 15‐kDa protein possessed conserved amino acid sequences of ADF of Toxoplasma gondii and Eimeria tenella. These characteristics indicate that the 15‐kDa protein of S. fayeri belongs to the ADF/cofilin protein family. The recombinant 15‐kDa protein evoked fluid accumulation in the looped ileum, resulting in diarrhea, but it did not kill the cultured fibroblast cells, macrophages or intestinal mucosal cells. In addition, the culture supernatant of the macrophages treated with the recombinant 15‐kDa protein killed the fibroblast L929 cells. This fact indicates that ADF of S. fayeri induced cytotoxic substances, such as tumor necrosis factor‐α, according to the published reports. Although further experiments are needed now to elucidate the enterotoxic mechanism of S. fayeris ADF, our findings may offer new insight into research on parasites and parasite‐instigated food poisoning.