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We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system. INTRODUCTIONMitosis, the process by which identical copies of the replicated genome are distributed to the products of each cell division, involves a highly dynamic sequence of coordinated motility events, mediated by a bipolar protein machine, the mitotic spindle (Karsenti and Vernos, 2001;Mitchison and Salmon, 2001;Gadde and Heald, 2004;Wadsworth and Khodjakov, 2004;Mogilner et al., 2006;Brust-Mascher and Scholey, 2007;Walczak and Heald, 2008). These motility events are driven by molecular-scale forces generated by mitotic kinesins and dyneins, together with dynamic microtubules (MTs), whose activities are controlled by a network of regulatory proteins, e.g., mitotic kinases, phosphatases, and proteolytic enzymes (Sharp et al., 2000c; BettencourtDias et al., 2004;Maiato and Sunkel, 2004;Rogers et al., 2005;Goshima et al., 2007). Among these mitotic proteins, the kinesin-5 motor is thought to play a key role (Cottingham et al., 1999;Valentine et al., 2006a;Civelekoglu-Scholey and Scholey, 2007).Purified kinesin-5 is a slow, modestly processive, plusend-directed bipolar homotetramer capable of cross-linking adjacent MTs and sliding apart antiparallel MTs in motility assays (Sawin et al., 1992;Cole et al., 1994;Kashina et al., 1996a;Kapitein et al., 2005;Tao et al., 2006;Valentine et al., 2006b;Krzysiak et al., 2008;Van den Wildenberg et al., 2008). In yeast cells the homotetrameric structure of kinesin-5 appears to be essential for mitosis (Hildebrandt et al., 2006), and in Drosophila embryos KLP61F displays dynamic properties consistent with an association with spindle MTs (Cheerambathur et al., 2008) ...

Helicobacter pylori has been suggested to play a role in the development of gastric carcinoma in humans. Also, mounting evidence indicates that cyclooxygenase-2 overexpression is associated with gastrointestinal carcinogenesis. We studied the effect of H. pylori on the expression and activity of cyclooxygenase-1 and cyclooxygenase-2 in MKN 28 gastric mucosal cells. H. pylori did not affect cyclooxygenase-1 expression, whereas cyclooxygenase-2 mRNA levels increased by 5-fold at 24 h after incubation of MKN 28 cells with broth culture filtrates or bacterial suspensions from wild-type H. pylori strain. Also, H. pylori caused a 3-fold increase in the release of prostaglandin E 2 , the main product of cyclooxygenase activity. This effect was specifically related to H. pylori because it was not observed with Escherichia coli and was independent of VacA, CagA, or ammonia. H. pylori isogenic mutants specifically lacking picA or picB, which are responsible for cytokine production by gastric cells, were less effective in the up-regulation of cyclooxygenase-2 mRNA expression and in the stimulation of prostaglandin E 2 release compared with the parental wild-type strain. This study suggests that development of gastric carcinoma associated with H. pylori infection may depend on the activation of cyclooxygenase-2-related events .Helicobacter pylori plays a central role in the etiology of chronic superficial gastritis and peptic ulcer disease and seems to increase the risk for development of gastric adenocarcinoma in humans (1-3). H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the release of a number of virulence factors such as urease, responsible for ammonia generation (4), a vacuolating cytotoxin (VacA) (5), and a cytotoxin-associated immunodominant protein (CagA) (6). In addition, multiple genes in the cag pathogenicity island have recently been described whose expression are necessary for cytokine production by gastric epithelial cells in vitro (7,8). However, the mechanism whereby H. pylori contributes to gastric carcinogenesis is still unknown.Prostaglandins (PGs) 1 are arachidonic acid derivatives that protect the gastric mucosa against exogenous injury (9, 10). PGs synthesis depends on the activity of a constitutively expressed and an inducible PG endoperoxide synthase/cyclooxygenase (COX-1 and COX-2, respectively) (10, 11). Mounting evidence indicates that COX-2 is associated with colorectal carcinogenesis (12, 13), COX-2 being overexpressed in 80 -90% of colorectal adenocarcinomas and in 40 -50% of premalignant adenomas (13). Moreover, COX-2 overexpression has recently been reported in human gastric adenocarcinoma (14). Although the role of COX-2 in gastrointestinal carcinogenesis is still unclear, its up-regulation is probably an early event (13).This study was designed to evaluate whether H. pylori affects COX-1 and COX-2 expression and PGE 2 synthesis in gastric mucosal cells (i.e. MKN 28 cells) (15, 16) in vitro and to study the role of H. pylori virulence factor...

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA+/VacA+ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.