Shinya Mitsuhashi

Allograft inflammatory factor-1 augments production of interleukin-6, -10and -12 by a mouse macrophage line

Mouse allograft inflammatory factor‐1 (AIF‐1) cDNA was cloned and the AIF‐1‐specific monoclonal antibodies were established to examine its tissue distribution. The mouse AIF‐1 was highly conserved among all reported AIF‐1 from a variety of species, from invertebrates to mammals, and the cloned cDNA was in good accordance with putative expressed regions of genomic sequences in the mouse major histocompatibility complex (MHC) class III region. The messages of mouse AIF‐1 were abundantly expressed in the testis, moderately in the spleen and lymph nodes and slightly in the liver and thymus of normal BALB/c mice. Immunohistological examination revealed that differentiating germ cells in the testis and presumably macrophages in the red pulp of the spleen were positive for AIF‐1. To analyse the function of the AIF‐1, a macrophage cell line, RAW 264.7, was transfected with mouse AIF‐1 cDNA. Upon stimulation with bacterial lipopolysaccharide, the transfectants that overexpressed AIF‐1 showed marked morphological changes and produced significantly large amounts of interleukin (IL)‐6, IL‐10 and IL‐12p40 but not IL‐12p70 compared with control cells. No difference was noted in production of tumour necrosis factor‐α, transforming growth factor‐β1 and IL‐1α. These results suggest that AIF‐1 plays an important role in cells of a monocyte/macrophage lineage upon stimulation with inflammatory stimuli by augmenting particular cytokine production.

The spiroketals containing a benzyloxymethyl moiety at C8 position showed the most potent apoptosis-inducing activity

The spiroketals containing a benzyloxymethyl moiety at the C8 position showed the most potent apoptosis-inducing activity, whereas its analogous compounds lacking any substituent at C8 or possessing ones other than the benzyloxymethyl moiety at C8 were all much less active. These results strongly suggest an important role of the benzyloxymethyl moiety linked to the C8 oxygen atom.

Nuclear Inhibitor of Protein Phosphatase-1 (NIPP1) Directs Protein Phosphatase-1 (PP1) to Dephosphorylate the U2 Small Nuclear Ribonucleoprotein Particle (snRNP) Component, Spliceosome-associated Protein 155 (Sap155) *□

Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Recent work has revealed essential roles for Ser/Thr phosphatases, such as protein phosphatase-1 (PP1), in splicing, but how these phosphatases are regulated is largely unknown. We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155 (spliceosome-associated protein 155), an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap155 dephosphorylation. C-terminally truncated NIPP1 (NIPP1-ΔC) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -ΔC resulted in slight and severe decreases in Sap155 hyperphosphorylation, respectively, and the latter was accompanied with inhibition of splicing. PP1 overexpression produced similar effects, whereas small interfering RNA-mediated NIPP1 knockdown enhanced Sap155 hyperphosphorylation upon okadaic acid treatment. NIPP1 did not inhibit but rather stimulated Sap155 dephosphorylation by PP1 in vitro through facilitating Sap155/PP1 interaction. Further analysis revealed that NIPP1 specifically recognizes hyperphosphorylated Sap155 thorough its Forkhead-associated domain and dissociates from Sap155 after dephosphorylation by associated PP1. Thus NIPP1 works as a molecular sensor for PP1 to recognize phosphorylated Sap155.

Pyrogallol Structure in Polyphenols is Involved in Apoptosis-induction on HEK293T and K562 Cells

As multiple mechanisms account for polyphenol-induced cytotoxicity, the development of structure-activity relationships (SARs) may facilitate research on cancer therapy. We studied SARs of representatives of 10 polyphenol structural types: (+)-catechin (1), (-)-epicatechin (2), (-)-epigallocatechin (3), (-)-epigallocatechin gallate (4), gallic acid (5), procyanidin B2 (6), procyanidin B3 (7), procyanidin B4 (8), procyanidin C1 (9), and procyanidin C2 (10). Amongst them, the polyphenols containing a pyrogallol moiety (3-5) showed the most potent cytotoxicic activity. These compounds evoked a typical DNA-laddering phenomenon in HEK293T, which indicated that the induction of apoptosis at least partly mediates their cytotoxic activity. Anti-oxidative capacity of compounds 3-5 were comparable to those of the trimers 9 and 10, which were not cytotoxic. Therefore, we suggest that pyrogallol moiety is important for fitting of polyphenols to their putative target molecule(s) in non-oxidative mechanism.

Protein phosphatase type 2A, PP2A, is involved in degradation of gp130.

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp130 at Ser-782 and degradation of gp130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp130. Taken together, present results strongly suggest that degradation of gp130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp130 is a potential therapeutic target in cancers. (Mol Cell Biochem 269: 183–187, 2005)

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells.

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.

Thyrsiferyl 23-acetate and its derivatives induce apoptosis in various T- and B-leukemia cells

Thyrsiferyl 23-acetate (TF-23A), a cytotoxic compound from marine red alga, induced a rapid cell death in various leukemic T- and B-cell lines. During incubation of Jurkat cells with TF-23A, condensation and fragmentation of nuclei occurred and clusters composed of uneven small cellular particles were formed. Concentration- and time-dependent DNA fragmentation was also induced by the incubation of the cells with TF-23A. These results demonstrate that the TF-23A-induced cell death follows a typical apoptotic process. The TF-23A-induced apoptosis was prevented by fetal calf serum and insulin, but not by EGF or PDGF. TF-23A and its several analogous compounds showed apoptosis-inducing activity. However, only TF-23A out of these compounds showed an inhibitory activity for protein phosphatase 2A, PP2A. These results strongly suggest that a structure of TF-23A involved in induction of apoptosis is different from that involved in the PP2A inhibition.

Protective effects of hesperidin derivatives and their stereoisomers against advanced glycation end-products formation

Context: Maillard reaction is implicated in the development of pathophysiology in age-related diseases. The search for newer Maillard reaction inhibitors is a priority among strategies to combat diabetes complications. Objective: To evaluate the inhibitory potential of hesperidin, its derivatives and their stereoisomers against advanced glycation end-products (AGEs) formation. Materials and methods: Hesperidin and hesperetin were chirally separated and the inhibitory effects of 1:1 mixture of (2S)- and (2R)-hesperidin (1), (2S)-hesperidin (2), (2R)-hesperidin (3), 1:1 mixture of (S)- and (R)-hesperetin (4), (S)-hesperetin (5), (R)-hesperetin (6), and monoglucosyl hesperidin (7) [1:1 mixture of (2S)-glucosyl hesperidin (8) and (2R)-glucosyl hesperidin (9)] at a concentration of 1 mM on protein glycation reaction have been revealed using the newly constructed RNase A-methylglyoxal (MGO) assay for the early stage and the bovine serum albumin (BSA)-glucose assay for the late stage of Maillard reaction. Results: This study has demonstrated that hesperidin and its derivatives possessed relatively strong activity against the formation of AGEs. (S)-Hesperetin (5) possessed the highest inhibitory rate up to 57.4% in BSA-glucose assay, 38.2% in RNase A-MGO assay. Discussion and conclusion: The new RNase A-MGO assay system could be used for the screening of AGEs inhibitors and hesperidin, and its derivatives could be promising candidate adjuvants for the treatment of diabetes complication, and age-related chronic diseases.

Quercetin inhibits advanced glycation end product formation via chelating metal ions, trapping methylglyoxal, and trapping reactive oxygen species

Physiological concentration of Mg2+, Cu2+, and Zn2+ accelerated AGE formation only in glucose-mediated conditions, which was effectively inhibited by chelating ligands. Only quercetin (10) inhibited MGO-mediated AGE formation as well as glucose- and ribose-mediated AGE formation among 10 polyphenols (1–10) tested. We performed an additional structure-activity relationship (SAR) study on flavanols (10, 11, 12, 13, and 14). Morin (12) and kaempherol (14) showed inhibitory activity against MGO-mediated AGE formation, whereas rutin (11) and fisetin (13) did not. These observations indicate that 3,5,7,4′-tetrahydroxy and 4-keto groups of 10 are important to yield newly revised mono-MGO adducts (16 and 17) and di-MGO adduct (18) having cyclic hemiacetals, while 3′-hydroxy group is not essential. We propose here a comprehensive inhibitory mechanism of 10 against AGE formation including chelation effect, trapping of MGO, and trapping of reactive oxygen species (ROS), which leads to oxidative degradation of 18 to 3,4-dihydroxybenzoic acid (15) and other fragments. Graphical abstract Inhibitory mechanism of quercetin against glucose-, ribose-, and MGO-mediated AGE formation and structures of MGO-quercetin adducts were elucidated.

MurA as a primary target of tulipalin B and 6-tuliposide B.

(−)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.