Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced ␣-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced ␣-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2,3-dialdehyde significantly abrogated toxin A-induced ␣-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced ␣-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates ␣-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.Clostridium difficile is the causative pathogen of antibioticassociated diarrhea and pseudomembranous colitis in humans and animals with a 10% symptomatic infection rate among hospitalized patients (1). Two toxins, A and B, released from C. difficile, are responsible for the massive fluid secretion, apoptosis of surface colonocytes, and acute enteritis seen during infection. The two exotoxins, which share ϳ63% amino acid homology, have glucosyltransferase activity (2-4) that inactivates Rho family proteins, leading to actin disaggregation (5, 6). Monoglucosylation of Rho, Rac, and Cdc42 by toxin A at threonine 37 prevents Rho family proteins from participating in the formation of actin filaments (7). This mechanism is believed to be a main cause for the cell rounding that is characteristic of toxinexposed cells (6,8). However, despite the presence of a rapid and severe change in the shape of infected cells, the effect of toxin A on the post-translational modification of tubulin and its subsequent influence on microtubule instability have not received detailed attention.Microtubule instability is critical to cell shape (9), cell movement (10), intracellular transport of organelles (11), and the separation of chromosomes during mitosis (12). This instability results in the continual and rapid turnover of microtubules, in a process that is crucial for cytoskeletal remodeling (9 -11). Because microtubules play a pivotal role in mitosis, drugs that infl...

Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the ␣-helical region of it (LLCIALRKK) that had antimicrobial activity (J. Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.-

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 μℳ), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nℳ, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.

Halogenated aliphatic compounds (HACs) can be reduced by iron sulfides in aqueous systems. Generally, the thermodynamics and kinetics of dehalogenation reactions are controlled by the mineralogical and particle surface characteristics of the iron sulfide, the composition of the HAC and reaction conditions such as component concentrations, pH and Eh. In this theoretical and experimental investigation of CCl 4 and C 2 Cl 6 reduction by FeS and FeS 2 , the roles of hydrophobic and hydrophilic sites on the iron sulfides were analyzed. Experimental data obtained through zeta potential measurements, were used along with the Gouy-Chapman model and the simple two-layer surface complexation model to relate iron sulfide surface hydroxyl densities to the degree of HAC dehalogenation. The surface hydroxyl site densities of FeS and FeS 2 were found to be 0.11 sites/nm 2 and 0.21 sites/nm 2 , respectively. During the dehalogenation reaction process, CCl 4 was found to decrease to its first intermediate product CHCl 3 within the first 20 hours followed by a slower process of conversion to CH 2 Cl 2 . The results also show that FeS is less hydrated (more hydrophobic) than FeS 2 . For CCl 4 and C 2 Cl 6 , FeS is a better dehalogenator than FeS 2 . These results imply that particle surface hydrophobicity is a critical factor in surface-mediated dehalogenation of chlorinated compounds.

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Antheraea yamamai silkworm cocoon is considered as a natural biocompatible materials, but can not be used as medical resources due to its difficult processability. In this study, we examined the general characteristics including cocoon shell weight and structural and thermal properties of Antheraea yamamai silkworm cocoon. The cocoon shell weight and thickness of wild silkworm cocoon was 0.528 g and 0.424 mm, respectively. The cocoon has yellow-green color in outside cocoon but white in inner layer cocoon. Amino acid analysis showed that the main amino acid of Antheraea yamamai cocoon is alanine, glycine, serine, aspartic acid, tyrosine and arginine. X-ray diffractometry showed that strong diffraction peaks at 2θ = 16. o C These results might be used as basic information for development of non-textile materials using Anthereae yamamai silkworm cocoon.

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The Epo/EpoR complex plays a critical role in the adhesion and migration of rat fibroblasts, and its functional inactivation is associated with PLC-γl-dependent reduction of cell-matrix adhesion and this also affects cell migration.