Replication fork collapse at a protein‐DNA roadblock leads to fork reversal,promoted by the RecQ helicase

Proteins that bind DNA are the cause of the majority of impediments to replication fork progression and can lead to subsequent collapse of the replication fork. Failure to deal with fork collapse efficiently leads to mutation or cell death. Several models have been proposed for how a cell processes a stalled or collapsed replication fork; eukaryotes and bacteria are not dissimilar in terms of the general pathways undertaken to deal with these events. This study shows that replication fork regression, the combination of replication fork reversal leading to formation of a Holliday Junction along with exonuclease digestion, is the preferred pathway for dealing with a collapsed fork in Escherichia coli. Direct endo‐nuclease activity at the replication fork was not observed. The protein that had the greatest effect on these fork processing events was the RecQ helicase, while RecG and RuvABC, which have previously been implicated in this process, were found to play a lesser role. Eukaryotic RecQ homologues, BLM and WRN, have also been implicated in processing events following replication fork collapse and may reflect a conserved mechanism. Finally, the SOS response was not induced by the protein‐DNA roadblock under these conditions, so did not affect fork processing.     

Echinacea purpurea L. in children: safety, tolerability, compliance, and clinical effectiveness in upper respiratory tract infections

Echinacea purpurea (L.) Moench was mistakenly taken from North America to Germany in 1939 where it was cultivated and various extractions were prepared and subsequently used to treat upper respiratory tract infections. Parents often administer Echinacea to their children, but safety data on the use of Echinacea in Canadian children is lacking. A screening history, physical examination, and daily record of symptoms from an initial visit through to a the follow-up visit 13 days later were used to increase patient safety. Each subject was administered an aerial part Echinacea extract. The dose was based on age (2.5 mL three times per day for children aged 2-5 years, and 5 mL two times per day for children aged 6-12 years) and administered for 10 days in an open-label trial. A rating scale was used to measure tolerance to the treatment. We assessed the safety and compliance of use of the Echinacea extract by measuring the amount of extract returned at the end of the study, having the parents complete and return a daily symptom diary, and recording the subjects' use of other natural health products or medications during the trial. Clinical effectiveness of the Echinacea extract could not be accurately assessed because of the small trial size and because the extract had been administered when some of the subjects had an upper respiratory tract infection that had begun 1 or more days prior to the study; however, each subject's symptoms improved. No allergic or adverse reaction occurred and no safety issues arose.     

Negative Regulation of TGFβ Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor—mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.     

Completion of the nucleotide sequence of the Enterococcus faecalis conjugative virulence plasmid pAD1 and identification of a second transfer origin

pAD1 is a 59.3-kb plasmid in Enterococcus faecalis that has been the subject of intense investigation with regard to its pheromone-inducible conjugation behavior as well as its contribution to virulence. Approximately two-thirds of the pAD1 nucleotide sequence has been previously reported. Here we report on an analysis of the final approximately 22 kb, a significant portion of which is believed to encode structural genes associated with conjugation. The conjugation-related region was also found to contain a new (second) origin of conjugative transfer (oriT). A list of open reading frames covering the entire plasmid is presented.     

The BiP Cochaperone ERdj4 Is Required for B Cell Development and Function

ERdj4 is a BiP cochaperone regulated by the unfolded protein response to facilitate degradation of unfolded and/or misfolded proteins in the endoplasmic reticulum. As the unfolded protein response plays a critical role in B cell maturation and antibody production, ERdj4 gene trap mice were generated to determine if this chaperone was required for B cell homeostasis. Homozygosity for the trapped allele resulted in hypomorphic expression of ERdj4 in bone marrow cells and abnormal development of hematopoietic lineages in the bone marrow. The number of myeloid cells was increased, while the number of erythroid and B lymphoid cells was reduced in ERdj4 gene trap mice compared to controls. An intrinsic B cell defect was identified that decreased survival of B cell precursors including large and small pre-B, and immature B cells. Consistent with impaired B lymphopoiesis, the number of mature follicular B cells was reduced in both the bone marrow and spleen of ERdj4 gene trap mice. Paradoxically, unchallenged ERdj4 gene trap mice showed non-specific hypergammaglobulinemia and gene trap B cells exhibited increased proliferation, survival and isotype switching in response to LPS stimulation. Although ERdj4 gene trap mice responded normally to T cell-independent antigen, they failed to mount a specific antibody response to T cell-dependent antigen in vivo. Collectively, these findings demonstrate that the chaperone activity of ERdj4 is required for survival of B cell progenitors and normal antibody production.     

Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.     

Hydroxymethylglutaryl coenzyme A reductase activity of adult Hymenolepis diminuta

The occurrence of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in adult Hymenolepis diminuta was demonstrated. This activity was negligible in the cestode's cytosolic fraction but was noted when the mitochondrial or microsomal fraction served as the enzyme source. The predominant localization of HMG-CoA reductase activity was with the microsomal fraction. This fraction did not contain appreciable mitochondrial contamination based on the distribution of marker enzymes. The enzymatic nature of HMG-CoA conversion to mevalonic acid by either fraction was apparent because the reaction was heat labile and responded linearly to time of assay and protein content. The enzymatic reduction of HMG-CoA absolutely required NADPH when either fraction was assayed. The lesser activity of the mitochondrial fraction was membrane-associated. The predominant localization of HMG-CoA reductase activity with microsomal membranes and its separation with the membranous component of the mitochondrial fraction suggest that mitochondrial activity reflects the presence of microsomal membranes. In its predominant localization and pyridine nucleotide requirement, the cestode's HMG-CoA reductase activity resembles that of mammalian systems. The finding of HMG-CoA reductase provides an enzymatic mechanism for the intermediate conversion of HMG-CoA to mevalonic acid that would be needed for acetate-dependent isoprenoid lipid synthesis by adult H. diminuta.     

Contrast-FEL—A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.