Eight new dimeric bromopyrrole alkaloids, nagelamides A-H (1-8) and a monomeric one, 9,10-dihydrokeramadine (9), have been isolated from the Okinawan marine sponge Agelas sp., and the structures were elucidated from spectroscopic data. Nagelamides A-H (1-8) exhibited antibacterial activity against Gram-positive bacteria. Nagelamide G (7) inhibited protein phosphatase 2A activity.

SUMMARYMouse allograft in¯ammatory factor-1 (AIF-1) cDNA was cloned and the AIF-1-speci®c 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 signi®cantly 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-a, transforming growth factor-b 1 and IL-1a. These results suggest that AIF-1 plays an important role in cells of a monocyte/macrophage lineage upon stimulation with in¯ammatory stimuli by augmenting particular cytokine production.

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Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 M TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.Protein phosphatases regulate numerous cellular functions and signal transduction pathways in cooperation with protein kinases (1, 2). Protein phosphatase types 1 and 2A, known as PP1 1 and PP2A, are two of four major protein serine/threonine phosphatases (PPs) that regulate diverse cellular events such as cell division, transcription, translation, muscle contraction, glycogen synthesis, and neuronal signaling (3-5).Okadaic acid (OA), a polyether fatty acid from the marine black sponge Halichondria okadai, was first identified as a small molecular weight inhibitor of PP and has been studied extensively (6). More than 40 compounds that inhibit PP1 as well as PP2A have been identified. Using these natural compounds, numerous experiments have been performed to analyze the roles of PPs in various cellular events (6, 7). The IC 50 values of such phosphatase inhibitors are almost identical for PP1 and PP2A, with the exception of compounds such as OA, TF-23A, and fostriecin (8 -10). PP2A is selectively inhibited by OA, TF-23A, and fostriecin, and this selectivity has made it possible to analyze PP2A function in living cells. However, no known inhibitor inhibits PP1 specifically. Oikawa et al. (11) reported the total chemical synthesis of tautomycin (TM), a small molecular weight PP inhibitor originally isolated from Streptomyces spiroverticillatus. Using the synthesized TM and related compounds, we previously examined the structure-function relationship of TM and found that the left-and right-hand moieties of TM are required for inhibition of PP and induction of apoptosis, respectively (12). We also re...

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-⌬C) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -⌬C 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.Pre-mRNA splicing is an essential step for expression of most genes in metazoans. Intron excision from a nascent transcript is achieved by pre-mRNA splicing catalyzed by the spliceosome, a macromolecular complex consisting of five small nuclear ribonucleoprotein particles (snRNPs) 4 and a large number of nonsnRNP proteins. Spliceosome assembly is an ordered process that includes stepwise recruitment of U1, U2, U5, and U4/6 snRNPs on a pre-mRNA and sequential formation of complex E 3 A/B 3 B* 3 C. The activated B* spliceosome catalyzes step I of splicing, whereas the C complex catalyzes step II. During and after splicing, spliceosome components dissociate and are recycled for further rounds of splicing. Spliceosome assembly/disassembly and splicing catalysis are thought to be regulated in part by reversible phosphorylation of spliceosomal proteins (1-3).U2 snRNP includes U2 snRNA and two heteromeric protein complexes, Sf3a and Sf3b. Sap155, also known as Sf3b1 or Sf3b155, is a component of the Sf3b and becomes hyperphosphorylated concomitant with or just after the first catalytic step of splicing in vitro (4). A recent study reveals that Sf3a/b proteins are destabilized and dissociate from the RNP core of the activated spliceosome during the transition from the B to C complex (5). Although Sf3a and Sf3b are essential early in the splicing reaction, they are apparently not required for the second catalytic step. Currently, it is not known what triggers exchange of proteins during spliceosome transitions. Shi et al. (6) reported that the protein Ser/Thr phosphatase (PPase) type 1 (PP1) and/or type 2A (PP2A) ar...

Physiological concentration of Mg, Cu, and Zn 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.

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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.