Subcellular localization determines the delicate balance between the anti- and pro-apoptotic activity of Livin

Livin is a member of the Inhibitor of Apoptosis Protein family which inhibits apoptosis induced by a variety of stimuli. We previously identified Livin and demonstrated that following apoptotic stimuli, Livin is cleaved by effector caspases to produce a truncated form with paradoxical pro-apoptotic activity. In the present study, we reveal that while full-length Livin shows diffuse cytoplasmic localization, truncated Livin (tLivin) is found in a peri-nuclear distribution with marked localization to the Golgi apparatus. Using mutation analysis, we identified two domains that are crucial for the pro-apoptotic activity of tLivin: the N-terminal region of tLivin which is exposed by cleavage, and the RING domain. We demonstrate that, of the N-terminal sequence, only the first N-terminal glycine residue dictates the peri-nuclear distribution of tLivin. However, while the perinuclear localization of tLivin is essential, it is not sufficient for tLivin to exert its pro-apoptotic function. Once tLivin is properly localized, an intact RING domain enables its pro-apoptotic function. Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

SslE Elicits Functional Antibodies That Impair In Vitro Mucinase Activity and In Vivo Colonization by Both Intestinal and Extraintestinal Escherichia coli Strains

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslE_IHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslE_IHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.     

Fluid secretory responses to enterotoxin STa and 8-bromo-cyclic GMP in fed and nutrionally-deprived gerbils: jejunum, ileum and colon in vivo

Fluid transport was measured gravimetrically in vivo in the jejunum, ileum and colon of fed, fasting (four days) and undernourished (50 % of control food intake for 21 days) gerbils (Gerbillus cheesmani). The effects of luminal enterotoxin Escherichia coli STa (50 ng/ml) and luminal 8-bromo-cyclic GMP (cGMP 1 mM) on fluid transport across jejunum, ileum and colon were also assessed. Fasting and undernourishment reversed the normal basal fluid absorption measured in fed ileum and colon into secretion. Neither fasting nor undernourishment had any effect on jejunal basal fluid absorption. In jejuna, ilea and colons of fed animals as well as in jejuna from fasting and undernourished gerbils STa (50 ng) reversed the normal absorptive "tone" to secretion but it had no significant effects on fluid secretion in either the ileum or colon from fasted gerbils. STa increased significantly the fluid secretion in ileum from undernourished gerbils. Luminal cGMP had no effect on basal absorptive tone in the jejunum of fed and fasted gerbils, but reversed absorption into secretion in the jejuna from undernourished gerbils. In the ilea taken from fed animals the small basal absorption was reversed to secretion by luminal cGMP. Although cGMP produced no significant changes in fluid secretion in the ilea taken from fasted gerbils, yet it caused a significant increase in those from undernourished gerbils. In the colon taken from fed animals cGMP decreased the basal fluid absorption significantly, but it had no significant effect on fluid secretion in the colon of fasted or undernourished gerbils. We conclude that fasting and undernourishment have no significant effects on fluid transport across the gerbil jejunum but reversed basal absorption in the fed ileum and colon into secretion. cGMP mimic the effects of STa in the jejunum taken from undernourished gerbils, in the ileum obtained under the three nutritional states and in the colon taken from fasting animals.     

Alcohol-triggered signs of migraine: An animal model

We describe an animal model where characteristics of migraine can be triggered by alcohol administration. In rats chronically implanted with a cannula overlying the transverse sinus, we applied potassium chloride (KCl) (or saline) to the meninges to sensitize trigeminovascular afferents. We assessed effects of repeated KCl application on animal behavior using conditioned place avoidance paradigm. In KCl-treated rats we discovered that alcohol injections (0.2?mg/kg), but not saline, resulted in the development of extracephalic allodynia and signs of ongoing pain.     

Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichia coli heat stable enterotoxin across small intestine of the gerbil (Gerbillus cheesmani)

The effects of mucosally added Escherichia coli heat stable enterotoxin (STa 30 ng ml(-1)) on the basal short-circuit current (Isc in microA cm(-2)) across stripped and unstripped sheets of jejuna and ilea taken from fed, starved (4 days, water ad lib) and undernourished (50% control food intake for 21 days) gerbil (Gerbillus cheesmani) were investigated. The effect of neurotoxin tetrodotoxin (TTX 10 microM) and the effects of replacing chloride by gluconate or the effects of removing bicarbonate from bathing buffers on the maximum increase in Isc induced by STa were also investigated. The maximum increase in Isc which resulted from the addition of STa were significantly higher in jejuna and ilea taken from starved and undernourished gerbils when compared with the fed control both using stripped and unstripped sheets. In the two regions of the small intestine taken from fed and starved animals TTX reduced the maximum increase in Isc induced by STa across unstripped sheets only. Moreover in jejuna and ilea taken from undernourished gerbils TTX reduced significantly the maximum increase in Isc induced by STa across stripped and unstripped sheets. Replacing chloride by gluconate decreased the maximum increase in Isc induced by STa across jejuna and ilea taken from undernourished gerbils only. Removing bicarbonates from bathing buffer decreased the maximum increase in Isc across the jejuna and ilea taken from starved and undernourished gerbils.