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These results identify K. septempunctata as the etiological agent of this novel food-borne illness outbreak associated with consumption of raw P. olivaceus. This is the first report, to our knowledge, demonstrating the human pathogenicity of Kudoa spores.

The ability of cancer cells to undergo invasion and migration is a prerequisite for tumor metastasis. Rho, a Ras-related small GTPase, and the Rho-associated coiled coil -containing protein kinases (Rho kinases, ROCK1 and ROCK2) are key regulators of focal adhesion, actomyosin contraction, and thus cell motility. Inhibitors of this pathway have been shown to inhibit tumor cell motility and metastasis. Here, we show that fasudil [1-(5-isoquinolinesulfonyl)-homopiperazine], an orally available inhibitor of Rho kinases, and its metabolite 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine) (fasudil-OH) modify tumor cell morphology and inhibit tumor cell migration and anchorage-independent growth. In addition, we show that fasudil inhibited tumor progression in three independent animal models. In the MM1 peritoneal dissemination model, tumor burden and ascites production were reduced by >50% (P < 0.05). In the HT1080 experimental lung metastasis model, fasudil decreased lung nodules by f40% (P < 0.05). In the orthotopic breast cancer model with MDA-MB-

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH 2 terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from ␤-naphthylamide derivatives and hydrolyzed citrulline-␤-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1, 2). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3-7). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH 2 -terminal Ca 2ϩ -binding protein of the S-100 family, linked to 10 -20 tandem filaggrin monomer repeats (8 -10). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2, 11-13). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(␥-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4, 6, 14, 15). Deimination is catalyzed by peptidylarginine deiminase (P...

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å . The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ␤-pore-forming toxins such as aerolysin, C. perfringens ⑀-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ␤-strands, two of which span its entire length. Domain II and domain III have three short ␤-strands each, by which they are distinguished. In addition, domain II has an ␣-helix lying on the ␤-strands. The sequence of amino acids composing the ␣-helix and preceding ␤-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ␤-barrel-made pores. These structural features imply that CPE is a ␤-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ␤-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins. Clostridium perfringens enterotoxin (CPE),2 which damages intestinal epithelia, is a causative agent of food poisoning. The toxin consists of a single chain polypeptide of 319 amino acids. The C-terminal domain of CPE (C-CPE, residues 184 -319) recognizes and binds to certain members of the claudin family, components of tight junctions, as a receptor on target cells (1-4), and the N-terminal region is believed to be involved in forming physiological pores to disrupt the selective permeability of the plasma membrane, resulting in cell death (5-7). It was also reported that the physiological pores are composed of a large complex comprising CPE and cellular components such as claudins (2,8).The binding between C-CPE and claudins has been well characterized. The 16 -17 C-terminal amino acids of C-CPE were reportedly important for the interaction (9, 10), especially, Tyr 306 , Tyr 310 , Tyr 312, and Leu 315 (11-13). According to the crystal structure of C-CPE (14), these residues organize a cleft space that is considered to interact directly with claudins. Claudins are tetratransmembrane proteins. The region of claudins responsible for binding to CPE was located on the C-terminal side of the second extracellular loop and recently designated CPE-SR for CPE sensitivity-related region (15). The bottom of the cleft space of CPE is negatively charged, whereas the CPESRs of CPE-sensitive claudins are positively charged. Therefore, it was proposed that electrostatic attraction at...

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