Exploiting vulnerabilities in the intracellular signaling pathways of tumor cells is a key strategy for the development of new drugs. The activation of cellular stress responses mediated by the endoplasmic reticulum (ER) allows cancer cells to survive outside their normal environment. Many proteins that protect cells against ER stress are active as protein disulfide isomerases (PDI) and the aim of this study was to test the hypothesis that apoptosis in response to ER stress can be increased by inhibiting PDI activity. We show that the novel chemotherapeutic drugs fenretinide and velcade induce ER stress-mediated apoptosis in melanoma cells. Both stress response and apoptosis were enhanced by the PDI inhibitor bacitracin. Overexpression of the main cellular PDI, procollagen-proline, 2-oxoglutarate-4-dioxygenase B subunit (P4HB), resulted in increased PDI activity and abrogated the apoptosis-enhancing effect of bacitracin. In contrast, overexpression of a mutant P4HB lacking PDI activity did not increase cellular PDI activity or block the effects of bacitracin. These results show that inhibition of PDI activity increases apoptosis in response to agents which induce ER stress and suggest that the development of potent, small-molecule PDI inhibitors has significant potential as a powerful tool for enhancing the efficacy of chemotherapy in melanoma. [Cancer Res 2008;68(13):5363-9]

C lostridium difficile infection (CDI) is a leading cause of pseudomembranous colitis and diarrhea (C. difficile-associated diarrhea [CDAD]) causally related to a perturbation of the intestinal microbiota due to antibiotic use. Although the transmission of CDI is primarily associated with health care and long-term care facilities, C. difficile is a ubiquitous microorganism that has been found in the environment. There are documented cases of community-acquired CDI; in fact, the community-acquired C. difficile infection rates in the United States have been reported to be 7.7 cases per 100,000 person-years, of which 35% were not associated with antibiotics (1). However, the rates associated with health care and long-term care facilities are much higher, possibly due to the colocalization of a reservoir of the pathogen and a high number of susceptible individuals housed in those environments (2). As the eradication of C. difficile spores is very difficult, spore reservoirs can persist within the health care and long-term care environment for long periods (3-6). In recent years, CDI has increased in severity and incidence, and part of this increase is due to the spread of epidemic antibiotic-resistant strains (7,8). Treatment options remain limited and even appear to be losing efficacy, as evidenced by the continued spread of the epidemic strain and increasing numbers of patients who experience relapses and recurrent disease (9).Clostridial species are normal members of the human gut flora, usually as a small fraction of the microbiome and mostly nontoxigenic species (10). C. difficile pathogenesis in humans is associated with the disruption of the normal enteric flora and colonization with a toxigenic C. difficile strain. This is followed by overgrowth of vegetative cells and production of toxins that damage the cells of the colon through enzymatic activity of a glucosyltransferase, which glucosylates cytoskeletal regulators, such as Ras and Rac (11). Toxigenic C. difficile strains produce at least one of the two major exotoxins, toxin A or toxin B, and most produce both. Only toxigenic strains have been shown to cause intestinal inflammatory and diarrheal disease (12, 13); therefore, toxins A and B are believed to be major virulence factors of CDI, although other lessstudied virulence components of the bacterium can contribute to the disease. For example, the presence of a third toxin known as binary toxin has been associated with a marked increase in disease severity and risk of death. This increase was seen in all strains carrying the gene for the binary toxin, not just the C. difficile NAP1/027 strain associated with recent virulent outbreaks (14), but it remains unclear whether the binary toxin itself causes in-

Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μℳ LPA in 60 μℳ Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mℳ Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway.

Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA measurements of pre-challenge sera showed that the median anti-toxin A and anti-toxin B IgG titres in the group of surviving animals were significantly higher than the median values in the group of animals that did not survive challenge. Assessment of the neutralizing activity of these sera revealed a statistically significant difference between the levels of both toxin A and toxin B neutralizing titres in protected versus unprotected animals as the median anti-toxin A and anti-toxin B neutralizing titres from surviving animals were higher than the median values from animals that succumbed to challenge. Statistically significant correlations between the toxin-specific binding titres and toxin neutralizing titres were seen for both toxin A and toxin B responses. The role of circulating anti-toxin antibodies in immunity against disease was evaluated by passive transfer of immune sera against C. difficile toxoids to naïve hamsters. Passively immunized animals were protected against morbidity and mortality associated with C. difficile challenge. Taken togeth...

Two winter barley (Hordeum vulgare L. cv. Igri) genomic clones, lambda gblt101.1 and lambda gblt101.2, encoding the blt101 gene family, were isolated from a genomic library. Deletion analysis of the blt101.1 promoter, using transient beta-glucuronidase (GUS) reporter expression assays, indicated that it contains at least three regulatory regions. A 107-bp region between nucleotides -168 and -275 with respect to the translation initiation codon, confers high-level GUS reporter expression at low temperature and contains a sequence (designated CR1) that is highly conserved in equivalent positions within the promoters of both members of the blt101 gene family. A 10-bp motif contained within CR1 binds proteins present in nuclear extracts from both control and low-temperature-treated barley tissue. Loss-of-function experiments, using transient-expression analysis, confirmed that this motif acts as a previously unreported low-temperature-responsive element. Nuclease sensitivity analysis of intact chromatin indicated that the blt101.1 promoter becomes more susceptible to DNase and micrococcal nuclease at low temperature, consistent with chromatin reorganisation upon transcriptional induction. It is proposed that both the 10-bp motif and chromatin reorganisation are involved in the regulation of blt101.1 at low temperature. This is the first detailed analysis of a low-temperature-specific plant promoter and identifies a novel low-temperature-response element.