Escherichia coli α-Hemolysin Counteracts the Anti-Virulence Innate Immune Response Triggered by the Rho GTPase Activating Toxin CNF1 during Bacteremia

The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1β signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1β antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1β and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1β secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.     

Gating defects of a novel Na+ channel mutant causing hypokalemic periodic paralysis

Hypokalemic periodic paralysis type 2 (hypoPP2) is an inherited skeletal muscle disorder caused by missense mutations in the SCN4A gene encoding the alpha subunit of the skeletal muscle Na+ channel (Nav1.4). All hypoPP2 mutations reported so far target an arginine residue of the voltage sensor S4 of domain II (R672/G/H/S). We identified a novel hypoPP2 mutation that neutralizes an arginine residue in DIII-S4 (R1132Q), and studied its functional consequences in HEK cells transfected with the human SCN4A cDNA. Whole-cell current recordings revealed an enhancement of both fast and slow inactivation, as well as a depolarizing shift of the activation curve. The unitary Na+ conductance remained normal in R1132Q and in R672S mutants, and cannot therefore account for the reduction of Na+ current presumed in hypoPP2. Altogether, our results provide a clear evidence for the role of R1132 in channel activation and inactivation, and confirm loss of function effects of hypoPP2 mutations leading to muscle hypoexcitability.     

A new peptidic vector for molecular imaging of apoptosis, identified by phage display technology

Phosphatidylserine (PS) exposure on the cell surface is an early marker of apoptosis. To select PS binding peptides as vectors of contrast agents to image apoptosis, a phage library has been exposed to perfused mouse livers. Phages not retained on control livers during the first perfusions were used for selections on apoptotic livers in a second series of perfusions. Four selected phages were further evaluated for binding to PS-coated enzyme-linked immunosorbent assay (ELISA) plates. They presented an apparent affinity constant (Ka app) for PS ranging from 6.08x10(10) M to 1.62x10(11)M. These phages did not bind to phosphatidylcholine, and competition with annexin V confirmed their specific interaction with PS. The phage with the highest affinity-bound PS in ELISA with a Ka app=(1.6+/-0.2)x10(11)M. It carried the TLVSSL peptide that was synthesized. Specific competition with annexin V and with the synthetic peptide was performed and confirms the specificity of the interaction.     

Myotonic dystrophy: size- and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism.

Myotonic dystrophy (DM) is a progressive neuromuscular disorder which results from elongations of an unstable (CTG)n repeat, located in the 3' untranslated region of the DM gene. A correlation has been demonstrated between the increase in the repeat number of this sequence and the severity of the disease. However, the clinical status of patients cannot be unambiguously ascertained solely on the basis of the number of CTG repeats. Moreover, the exclusive maternal inheritance of the congenital form remains unexplained. Our observation of differently sized repeats in various DM tissues from the same individual may explain why the size of the mutation observed in lymphocytes does not necessarily correlate with the severity and nature of symptoms. Through a molecular and genetic study of 142 families including 418 DM patients, we have investigated the dynamics of the CTG repeat meiotic instability. A positive correlation between the size of the repeat and the intergenerational enlargement was observed similarly through male and female meioses for < or = 0.5-kb CTG sequences. Beyond 0.5 kb, the intergenerational variation was more important through female meioses, whereas a tendency to compression was observed almost exclusively in male meioses, for > or = 1.5-kb fragments. This implies a size- and sex-dependent meiotic instability. Moreover, segregation analysis supports the hypothesis of a maternal as well as a familial predisposition for the occurrence of the congenital form. Finally, this analysis reveals a significant excess of transmitting grandfathers partially accounted for by increased fertility in affected males.     

A Starling resistor regulates cerebral venous outflow in dogs

This investigation was undertaken to determine whether a Starling resistor or venous waterfall effect exists between the sagittal sinus and the cerebral veins such that increases in sagittal sinus pressure (Pss) do not abolish cerebral venous outflow and to examine two possible contributions of extracranial venous valves in regulating outflow. Anesthetized dogs were subjected to positive end-expiratory pressure (PEEP) before and after intracranial pressure (Pic) was elevated by inflation of an epidural balloon. PEEP raised Pss equally in all animals, but Pic and cerebral venous pressure (Pcv) increased less in the presence of intracranial hypertension. When Pss was low, passage of a catheter in the cerebral vein in and out of the sagittal sinus demonstrated an abrupt drop in pressure as the sinus was entered. When Pss was raised and lowered independently of superior vena caval pressure (Psvc) the changes in Pic and Pcv were less when Pss was decreased than when it was increased. Sustained increases and decreases in Psvc caused increases and decreases in Pcv, Pic, Pss, and external jugular venous pressure (Pejv) regardless of whether external jugular venous valves were present or absent. We conclude that a Starling resistor between the sagittal sinus and the cerebral veins regulates cerebral venous outflow when Pss is increased by PEEP and other maneuvers that raise Psvc. The waterfall maintains Pcv and Pic at normal levels when Psvc and Pss are reduced. Extracranial venous valves are not essential to this mechanism.     

Significance of ecological vicariance and long-distance dispersal in the diversification of Carex sect. Spirostachyae (Cyperaceae)

Plant disjunctions have provided one of the most intriguing distribution patterns historically addressed by biogeographers. Carex sect. Spirostachyae (Cyperaceae) displays an interesting pattern of disjunction to evaluate these scenarios, with species occurring in the main continental landmasses and in oceanic islands of the two hemispheres. Internal transcribed spacer and 5'-trnK intron plastid gene sequences were analyzed to determine (1) the times of diversification using penalized likelihood, and (2) reconstructions of the regions using maximum likelihood and Bayesian approaches of origin of sect. Spirostachyae and internal main lineages. The times for the diversification of sect. Spirostachyae are dated to between the end of the Eocene and the Oligocene, whereas the two main lineages are dated to between the end of the Oligocene and the beginning of Miocene. The phylogenetic analyses reveal a Mediterranean-Eurasian center of differentiation for sect. Spirostachyae and subsection Spirostachyae, whereas no clear, single ancestral area could be inferred for subsection Elatae. Both long-distance dispersal and ecological vicariance appear to have been involved in the evolutionary history of the disjunct distribution of the main lineages of sect. Spirostachyae. These organisms appear to have a special ability to colonize remote areas (through transoceanic and interhemispherical colonizations), but special long-distance dispersal mechanisms are not evident.     

The accuracy of protein synthesis in reticulocyte and HeLa cell lysates

The accuracy of translation in protein synthesis is measured as the rate of misincorporation of a particular amino acid, different from that specified by an mRNA codon, into protein. The cowpea variant of tobacco mosaic virus, CcTMV, contains no cysteine or methionine in its coat protein. Translation in vitro of purified CcTMV coat protein mRNA by rabbit reticulocyte and HeLa cell lysates has been performed. The coat protein product was purified by immunoprecipitation with specific antisera, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The error rate was measured by comparing the incorporation of [35S]cysteine with incorporation of [3H]leucine, and the total CcTMV coat protein synthesized was calculated from its known leucine content. An error rate of (1-2) X 10(-3) cysteines/CcTMV coat protein was obtained with reticulocyte lysates. If errors were cysteine incorporation in place of arginine, this number is converted to 3 X 10(-4) cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9 X 10(-6) cysteines/amino acid. The error frequencies with HeLa cell lysates were 6-fold higher. Paromomycin, a eukaryotic misreading antibiotic, increased error rates 10-fold in both lysates. These data compare well with in vivo measurements and suggest that some transformed cells may survive with higher mistranslation rates.