Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in the signal transduction of various cellular responses. However, although it is undeniably important that modulators of PLC activity be identified, no direct PLC activity modulator has been identified until now. In this study, by screening more than 10,000 different compounds in human neutrophils, we identified a compound that strongly enhances superoxide-generating activity, which is well known to be PLC-dependent. The active compound 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide (m-3M3FBS) stimulated a transient intracellular calcium concentration ([Ca 2ϩ ] i ) increase in neutrophils. Moreover, m-3M3FBS stimulated the formation of inositol phosphates in U937 cells, indicating that it stimulates PLC activity. The compound showed no cell-type specificity in terms of [Ca 2ϩ ] i increase in the various cell lines including leukocytes, fibroblasts, and neuronal cells. We also ruled out the possible involvement of heterotrimeric G proteins in m-3M3FBS-stimulated signaling by confirming the following: 1) pertussis toxin does not inhibit m-3M3FBS-induced [Ca 2ϩ ] i increase; 2) m-3M3FBS does not stimulate cyclic AMP generation; and 3) the inhibition of G q by the regulator of G proteinsignaling 2 does not affect the m-3M3FBS-induced [Ca 2ϩ ] i increase. We also observed that m-3M3FBS stimulated PLC activity in vitro. The purified isoforms of PLC that were tested (i.e., ␤2, ␤3, ␥1, ␥2, and ␦1) were activated by m-3M3FBS and showed no isoform specificity. Taken together, these results demonstrate that m-3M3FBS modulates neutrophil functions by directly activating PLC. Because m-3M3FBS is the first compound known to directly activate PLC, it should prove useful in the study of the basic molecular mechanisms of PLC activation and PLC-mediated cell signaling.

This review focuses on the expanding role of marine collagen (MC)-based scaffolds for biomedical applications. A scaffold—a three-dimensional (3D) structure fabricated from biomaterials—is a key supporting element for cell attachment, growth, and maintenance in 3D cell culture and tissue engineering. The mechanical and biological properties of the scaffolds influence cell morphology, behavior, and function. MC, collagen derived from marine organisms, offers advantages over mammalian collagen due to its biocompatibility, biodegradability, easy extractability, water solubility, safety, low immunogenicity, and low production costs. In recent years, the use of MC as an increasingly valuable scaffold biomaterial has drawn considerable attention from biomedical researchers. The characteristics, isolation, physical, and biochemical properties of MC are discussed as an understanding of MC in optimizing the subsequent modification and the chemistries behind important tissue engineering applications. The latest technologies behind scaffold processing are assessed and the biomedical applications of MC and MC-based scaffolds, including tissue engineering and regeneration, wound dressing, drug delivery, and therapeutic approach for diseases, especially those associated with metabolic disturbances such as obesity and diabetes, are discussed. Despite all the challenges, MC holds great promise as a biomaterial for developing medical products and therapeutics.

The development of Ag-presenting functions by murine dendritic cells (DCs) of the CD8+ DC lineage was studied using a Flt-3 ligand stimulated bone-marrow culture system. Although newly formed DCs of this lineage are capable of Ag uptake and efficient presentation to T cells on MHC class II, they initially lack the ability to cross-present exogenous Ags on MHC class I. Cross-presentation capacity is acquired as a subsequent maturation step, promoted by cytokines such as GM-CSF. The development of cross-presentation capacity by the DCs in these cultures may be monitored by the parallel development of DC surface expression of CD103. However, the expression of CD103 and cross-presentation capacity are not always linked; therefore, CD103 is not an essential part of the cross-presentation machinery. These results explain the considerable variability in CD103 expression by CD8+ DCs as well as the findings that not all DCs of this lineage are capable of cross-presentation.

The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. Here we examined the effect of ascochlorin, a prenyl-phenol anti-tumor compound from the fungus Ascochyta viciae, on the regulation of signaling pathways that control MMP-9 expression in human renal carcinoma (Caki-1) cells. Ascochlorin reduced the invasive activity of Caki-1 cells and inhibited phorbol 12-myristate 13-acetate-induced increases in MMP-9 expression and activity in a dose-dependent manner. Reporter gene, electrophoretic mobility shift, kinase inhibitor assays, and in vitro kinase assay showed that ascochlorin inhibits MMP-9 gene expression by suppressing activation of the nuclear transcription factor activator protein-1 (AP-1) via the extracellular signalregulated kinase 1 and 2 pathway. The AP-1 family member most specifically affected by ascochlorin was Fra-1. Ascochlorin did not affect the activation of the c-Jun N-terminal or p38 kinase pathways. Moreover, transfection of Caki-1 cells with AP-1 decoy oligodeoxynucleotides resulted in the suppression of phorbol 12-myristate 13-acetate-induced MMP-9 expression and invasion. In conclusion, ascochlorin represents a unique natural anti-tumor compound that specifically inhibits MMP-9 activity through suppression of AP-1-dependent induction of MMP-9 gene expression.Ascochlorin (see Fig. 1A), a prenyl-phenol compound isolated from the fungus, Ascochyta viciae, was originally found to have antiviral antibiotic activity (1). In addition to its antiviral and anti-fungal activity, natural and synthetic derivatives of ascochlorin reduce serum cholesterol and triglyceride levels suppress hypertension and tumor development and ameliorate type I and II diabetes (2-9). Moreover, several ascochlorin derivatives have been reported to be potent agonists of nuclear hormone receptors, including peroxisome proliferator-activated receptor ␥, suggesting that the structure of ascochlorin would be useful in designing modulators of nuclear receptors (6, 10, 11, 13).The most well known extracellular matrix (ECM) 1 -degrading enzymes are the matrix metalloproteinases (MMPs). MMPs are a family of zinc-dependent endoproteinases that are capable of degrading all the components of the ECM. MMPs are structurally and functionally homologous proteins, with at least 20 known members. MMPs can be divided into four families based on structure and substrate specificity: collagenases, gelatinases, stromelysins, and membrane-associated MMPs. Among the previously reported human MMPs, gelatinase-A (MMP-2) and gelatinase-B (MMP-9) are key enzymes that degrade type IV collagen (14, 15). These two gelatinases share structural and catalytic similarities, but transcription of the MMP-2 and MMP-9 genes is independently regulated due to distinct arrays of regulatory elements in the gene promoters. Regulated expression of MMP-9 has been implicated in renal development, macrophage differentiation, atherosclerosis, inflammation, rheumatoid arthritis, and tumor invasion (16 -17). I...

The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in the invasion and metastasis of cancer cells. Here, we found that an antitumor antibiotic, ascofuranone, inhibits invasion and MMP-9 induction induced by phorbol myristate acetate (PMA) in human cell lines. Ascofuranone also inhibits the protein expression and transcription of MMP-9 induced by tumor necrosis factor-alpha. The inhibition of MMP-9 induction was observed in human cancer cell lines as well as primary rat mesangial cells. Furthermore, as evidenced by MMP-9 promoter and electrophoretic mobility shift assays, ascofuranone specifically inhibited MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, ascofuranone suppressed PMA-induced phosphorylation of Ras, Raf, MEK and extracellular signal-regulated kinase (ERK), upstream factors involved in AP-1activation, whereas the phosphorylation of p38 and JNK/mitogen-activated protein kinase was not affected by ascofuranone, suggesting that the primary target of ascofuranone for suppression of the AP-1 induction is present in upstream of ERK signaling pathway. These results suggest that the suppression of MMP-9 expression, at least in part, contributes to the antitumor activity of ascofuranone.

No abstract

Purpose: Recent studies have shown that 15-deoxy-Δ 12, 14 -prostaglandin J 2 (15d-PGJ 2 ), a natural ligand for peroxisome proliferator-activated receptor-γ (PPARγ), inhibits cell proliferation and induces apoptosis. The specific molecular mechanisms underlying this effect remain to be elucidated. We examined whether 15d-PGJ 2 has antitumor activity in vitro and in vivo, and investigated the underlying mechanism. Experimental Design: We examined 15d-PGJ 2 -induced apoptosis in human leukemia cells in the context of mitochondrial injury, oxidative damage, and signaling pathway disturbances. In addition, we investigated the antitumor effect of 15d-PGJ 2 in a mouse CT-26 s.c. tumor model and HL-60 leukemia xenograft model. Results: 15d-PGJ 2 induced apoptosis in leukemia and colorectal cancer cells in a dosedependent manner and led to generation of reactive oxygen species (ROS) through mitochondria and NADPH oxidase activation, activation of JNK, and inactivation of Akt, a serine/threonine-specific protein kinase. Constitutive activation of Akt for an engineered myristoylated protein prevented 15d-PGJ 2 -mediated apoptosis but not ROS generation. Collectively, these findings suggest a hierarchical model of apoptosis induced by 15d-PGJ 2 in human leukemia cells: oxidative injury represents a primary event resulting in Akt inactivation, which in turn leads to mitochondrial injury and apoptosis. Moreover The peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor belonging to the nuclear receptor superfamily (1, 2) and is expressed in some myeloid leukemic cell lines (3). PPARγ agonists include the natural ligands 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ; refs. 4, 5) and lysophosphatidic acid (6), and the synthetic thiazolidinedione class of antidiabetic drugs such as ciglitazone (7). The cyclopentenone PG 15d-PGJ 2 is a product of the cyclooxygenase pathway and is the final metabolite of PGD 2 (8). 15d-PGJ 2 has highly reactive structures that contain an α, β-unsaturated ketone susceptible to nucleophilic addition reactions. For example, the cyclopentenone ring of 15d-PGJ 2 covalently modifies cellular proteins such as the p65 and p50 subunits of NF-κB (9, 10). The potent antiproliferative and antiviral effects of cyclopentenone PGs are attributed to this reactivity (11).15d-PGJ 2 has recently received attention because it functions as a potential regulator of diverse processes, including cell growth, differentiation, and inflammation. It also exerts antitumorigenic activity; for example, 15d-PGJ 2 activation of PPARγ significantly inhibits cell growth and induces apoptosis in several types of cancer cells (12)(13)(14)(15). Some effects of 15d-PGJ 2 are exerted through its interaction with PPARγ (4, 5); however, more recent evidence indicates that 15d-PGJ 2 can also act independently of PPARγ (16). Until now, the molecular mechanism of the antineoplastic activity of 15d-PGJ 2 has not been fully elucidated. In addition, controversial data on the role of PPARγ in colon cancer sugg...

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of antiwall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.