Many lines of evidence suggest that a-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of a-synuclein, and the mechanistic features of the phenomenon. We first showed that not only a-synuclein but also b-and c-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of a-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. a-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of a-synuclein at 4°C was comparable with the import observed at 37°C. Furthermore, membrane translocation of a-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of a-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-L: -cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.

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.-

The 18 F-FDG uptake pattern on PET could be an indicator of the prognosis and aggressiveness of various tumors, including hepatocellular carcinoma (HCC). Hexokinase, especially hexokinase type II (HKII), plays a critical role in 18 F-FDG uptake in rapidly growing tumors. We established a stable cell line overexpressing HKII by the transfection of full DNA of HKII to HCC cells (SNU449) that express low levels of HKII and investigated how 18 F-FDG uptake mechanisms, especially overexpression of HKII, are linked to tumor proliferation mechanisms. Methods: The HKII gene was stably transfected to SNU449 cells with an expression vector. HKII expression in the cells was verified by reverse-transcriptase polymerase chain reaction, Western blot analysis, adenosine triphosphate (ATP) and lactate production, 18 F-FDG uptake measurement, and confocal microscopy. Cellular proliferation activity and response to the anticancer drug cisplatin were evaluated by cell counting using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For the evaluation of molecular pathways involved in tumor proliferation, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was investigated. Results: The stable cell line produced HKII effectively, but expression of other enzymes or transporters for glycolysis, such as glucose-6-phosphatase (G6Pase), HKI and III, and glucose transporter type 1 and 2 (Glut-1 and Glut-2), did not show any changes. 18 F-FDG uptake was significantly increased after transfection. ATP and lactate production was also increased after transfection. Overexpressed HKII was associated with mitochondria on confocal microscopy. Cells with overexpression of HKII, compared with the nontransfected cell line, showed 1.5-to 2-fold higher cell survival and resistance to the anticancer agent cisplatin (2-to 8-fold). In the molecular study, the activated form of Akt was increased after transfection, and PI3K inhibitor dissociated the mitochondrial HKII to the cytoplasm. In addition, the adenosine monophosphate-activated protein kinase (AMPK) pathway is also involved in Akt signaling. Conclusion: HKII plays an important role in 18 F-FDG uptake and tumor proliferation by both the PI3K-dependent and the PI3K-independent Akt signal pathways; therefore, the 18 F-FDG uptake pattern on a PET scan can be a surrogate marker of prognosis in HCC.

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Although previous studies on hexokinase (HK) II indicate both the N-and C-terminal halves are catalytically active, we show in this study the N-terminal half is significantly more catalytic than the C-terminal half in addition to having a significantly higher Km for ATP and Glu. Furthermore, truncated forms of intact HK II lacking its first N-terminal 18 amino acids (Δ18) and a truncated N-terminal half lacking its first 18 amino acids (Δ18N) have higher catalytic activity than other mutants tested. Similar results were obtained by PET-scan analysis using

Behçet's disease (BD) is a chronic, multisystemic vasculitis that theoretically affects all sizes and types of blood vessels. Although pathogenesis remains enigmatic, endothelial cells are believed to be the primary target in this disease. We detected the target protein using western blotting and immunoprecipitation and determined the amino-acid sequence of the peptide by liquid chromatography-matrix assisted laser desorption/ionization-tandem time-of-flight analysis (LC-MALDI-TOF/TOF). Serum reactivity against the recombinant target protein was analyzed by immunoblotting. Serum reactivity against streptococcal 65-kD heat shock protein (hsp-65) and the recombinant target protein was investigated by ELISA. The 36-40-kD protein band that was obtained from immunoprecipitation, which was analyzed by LC-MALDI-TOF/TOF, exhibited the amino-acid sequences of heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP-A2/B1). Reactivity of serum IgA against human recombinant hnRNP-A2/B1 was detected in 25 of 30 BD patients (83.3%), 4 of 30 systemic lupus erythematosus patients (13.3%), 8 of 30 rheumatoid arthritis patients (26.7%), 9 of 30 Takayasu's arteritis patients (30%), 6 of 30 healthy controls (20%), and none of 30 IgA nephropathy patients. Optical densities obtained from ELISAs against the recombinant human hnRNP-A2/B1 were correlated with those against the recombinant streptococcal hsp-65.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.