Hemophagocytic macrophages harbor Salmonella enterica during persistent infection

Salmonella enterica subspecies can establish persistent, systemic infections in mammals, including human typhoid fever. Persistent S. enterica disease is characterized by an initial acute infection that develops into an asymptomatic chronic infection. During both the acute and persistent stages, the bacteria generally reside within professional phagocytes, usually macrophages. It is unclear how salmonellae can survive within macrophages, cells that evolved, in part, to destroy pathogens. Evidence is presented that during the establishment of persistent murine infection, macrophages that contain S. enterica serotype Typhimurium are hemophagocytic. Hemophagocytic macrophages are characterized by the ingestion of non-apoptotic cells of the hematopoietic lineage and are a clinical marker of typhoid fever as well as certain other infectious and genetic diseases. Cell culture assays were developed to evaluate bacterial survival in hemophagocytic macrophages. S. Typhimurium preferentially replicated in macrophages that pre-phagocytosed viable cells, but the bacteria were killed in macrophages that pre-phagocytosed beads or dead cells. These data suggest that during persistent infection hemophagocytic macrophages may provide S. Typhimurium with a survival niche.     

Repeated bouts of eccentrically biased endurance exercise stimulate salivary IgA secretion rate

To determine the salivary secretory immunoglobulin A (sIgA) response to repeated bouts of unaccustomed, downhill running (eccentrically biased) and examine potential protective immunological adaption from a repeated bout effect. Eleven active but untrained males (age: 19.7±0.4 years; VO_2peak: 47.8± 3.6 ml · kg⁻¹ · min ⁻¹) performed two 60 min bouts (Run 1 and Run 2) of downhill running (−13.5% gradient), separated by 14 days, at a speed eliciting 75% of their VO_2peak on a level grade. Saliva samples were collected before (baseline), immediately post exercise (IPE), and every hour for 12 h and every 24 h for 6 days after each run. Salivary sIgA concentration was measured and sIgA secretion rate was calculated. Results were analysed using repeated measures ANOVA (12 h period: 2x14; 24 h intervals: 2x7; p ≤ 0.05) with Tukey post-hoc tests where appropriate. Results are reported as means ± SE. There was a significant (p < 0.0001) interaction effect for sIgA secretion rate, IPE, with higher values after Run 2, as well as a significant (p < 0.01) time effect with elevated levels IPE and between 24 h and 144 h. There was a run effect (p < 0.0001), with the sIgA secretion rate significantly higher after Run 2. Repeated bouts of unaccustomed, eccentrically biased exercise induced alterations in the salivary sIgA secretion rate. This may serve as a protective mucosal adaptation to exercise-induced tissue damage.     

NH2-terminal Deletion of β-Catenin Results in Stable Colocalization of Mutant β-Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion

β-Catenin is essential for the function of cadherins, a family of Ca²⁺-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH₂-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH₂-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH₂-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH₂-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH₂ terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.     

Patterns of extensive genetic differentiation and variation among European harbor seals (Phoca vitulina vitulina) revealed using microsatellite DNA polymorphisms

The harbor seal (Phoca vitulina) has the most extensive distribution of any phocid seal species. An analysis of population structure in this species across its European range was made using 7 phocid derived microsatellites in a sample of 1,029 individuals from 12 separate geographic areas. Despite the species potential for long-distance movement, significant genetic differentiation between areas was observed using an unbiased estimator of RST. Six distinct population units were identified: Ireland-Scotland, English east coast, Waddensea, western Scandinavia (Norway-Kattegat-Skagerrak-west Baltic), east Baltic, and Iceland. Little local substructuring is present along coastlines with a continuous distribution of breeding animals, but differentiation does increase with geographic distance. The degree of differentiation is greater over equivalent distances where the distribution is discontinuous, such as along coasts where breeding colonies are separated by large distances or by stretches of open sea. Patterns of population differentiation derived from microsatellites are very similar to those obtained from previous mitochondrial DNA analysis and suggest that philopatry in harbor seals operates over 300-500 km. In Europe, harbor seals have experienced a complex demographic history and patterns of population structure are likely to have been affected by natural environmental influences such as Pleistocene glaciations and epizootics. Comparison of Nm values from an unbiased estimator of RST, GST, and theta are consistent and, in some cases, may indicate populations where conditions deviate from the expectations of the RST model.      

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.     

A panoramic view of yeast noncoding RNA processing

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.     

Network crosstalk dynamically changes during neutrophil polarization

How complex signaling networks shape highly coordinated, multistep cellular responses is poorly understood. Here, we made use of a network-perturbation approach to investigate causal influences, or "crosstalk," among signaling modules involved in the cytoskeletal response of neutrophils to chemoattractant. We quantified the intensity and polarity of cytoskeletal marker proteins over time to characterize stereotyped cellular responses. Analyzing the effects of network disruptions revealed that, not only does crosstalk evolve rapidly during polarization, but also that intensity and polarity responses are influenced by different patterns of crosstalk. Interestingly, persistent crosstalk is arranged in a surprisingly simple circuit: a linear cascade from front to back to microtubules influences intensities, and a feed-forward network in the reverse direction influences polarity. Our approach provided a rational strategy for decomposing a complex, dynamically evolving signaling system and revealed evolving paths of causal influence that shape the neutrophil polarization response.     

Activation of JNK by Epac is independent of its activity as a Rap guanine nucleotide exchanger

Guanine nucleotide exchange factors (GEFs) and their associated GTP-binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified Epac (exchange protein activated by cAMP) as a strong activator of JNK-1. Epac is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily of Ras small G-proteins. We report here that while Epac activates the JNK severalfold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce Epac-dependent JNK activation. Moreover, Epac signaling to the JNKs was not blocked by inactivation of endogenous Rap, suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for Epac-mediated activation of JNK. These studies identified a region overlapping the Ras exchange motif domain as critical for JNK activation. Consistent with this, an isolated Ras exchange motif domain from Epac is sufficient to activate JNK. We conclude that Epac signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by Epac.     

Enteroid Monolayers Reveal an Autonomous WNT and BMP Circuit Controlling Intestinal Epithelial Growth and Organization

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