Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

Activation of muscle-specific receptor tyrosine kinase and binding to dystroglycan are regulated by alternative mRNA splicing of agrin

Agrin induces the aggregation of postsynaptic proteins at the neuromuscular junction (NMJ). This activity requires the receptor-tyrosine kinase MuSK. Agrin isoforms differ in short amino acid stretches at two sites, called A and B, that are localized in the two most C-terminal laminin G (LG) domains. Importantly, agrin isoforms greatly differ in their activities of inducing MuSK phosphorylation and of binding to alpha-dystroglycan. By using site-directed mutagenesis, we characterized the amino acids important for these activities of agrin. We find that the conserved tripeptide asparagineglutamate-isoleucine in the eight-amino acid long insert at the B-site is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. We also show that binding to alpha-dystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding.     

Peroxisome biogenesis: where Arf and coatomer might be involved

The present review summarizes recent observations on binding of Arf and COPI coat to isolated rat liver peroxisomes. The general structural and functional features of both Arf and coatomer were considered along with the requirements and dependencies of peroxisomal Arf and coatomer recruitment. Studies on the expression of mammalian Pex11 proteins, mainly Pex11alpha and Pex11beta, intimately related to the process of peroxisome proliferation, revealed a sequence of individual steps including organelle elongation/tubulation, formation of membrane and matrix protein patches segregating distinct proteins from each other, development of membrane constrictions and final membrane fission. Based on the similarities of the processes leading to cargo selection and concentration on Golgi membranes on the one hand and to the formation of peroxisomal protein patches on the other hand, an implication of Arf and COPI in distinct processes of peroxisomal proliferation is hypothesized. Alternatively, peroxisomal Arf/COPI might facilitate the formation of COPI-coated peroxisomal vesicles functioning in cargo transport and retrieval from peroxisomes to the ER. Recent observations suggesting transport of Pex3 and Pex19 during early steps of peroxisome biogenesis from the ER to peroxisomes inevitably propose such a retrieval mechanism, provided the ER to peroxisome pathway is based on transporting vesicles.     

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.     

Frequency of local, regional, and long-distance dispersal of diploid and tetraploid Saxifraga oppositifolia (Saxifragaceae) to Arctic glacier forelands

? Premise of the Study: Climate change forces many species to migrate. Empirical small-scale data on migration and colonization in the Arctic are scarce. Retreating glaciers provide new territory for cold-adapted plant species, but the genetic consequences depend on dispersal distances and frequencies. We estimated local, regional, and long-distance dispersal frequencies, as well as their effect on levels of genetic diversity, in diploid and tetraploid individuals of Saxifraga oppositifolia. ? Methods: Samples were collected in four aged moraines in each of three glacier forelands, in surrounding areas and reference populations in the Arctic archipelago Svalbard. These samples were analyzed for neutral amplified fragment length polymorphisms (AFLPs, n = 707) and ploidy levels (n = 30). ? Key Results: Genetic clustering and ploidy analyses revealed two distinct genetic groups representing diploids and tetraploids, with few intermediate triploids. The groups were intermixed in most sampled populations. No differences in genetic diversity were found between tetraploids and diploids, or between established and glacier foreland populations. Seeds were dispersed over local, regional, and long distances, with the highest proportions of seeds originating from close sources. A minimum of 4-15 founding individuals from several source populations had initially established in each glacier foreland. ? Conclusions: Our data suggest that S. oppositifolia can rapidly colonize new deglaciated areas without losing genetic diversity. Thus, glacier forelands can be alternative habitats for cold-adapted vascular plants tracking their climatic niche. Our data show no difference in colonization success between diploid and tetraploid individuals.     

Lower Prevalence of Carotid Plaque Hemorrhage in Women,and Its Mediator Effect on Sex Differences in Recurrent Cerebrovascular Events

Background and Purpose

Women are at lower risk of stroke, and appear to benefit less from carotid endarterectomy (CEA) than men. We hypothesised that this is due to more benign carotid disease in women mediating a lower risk of recurrent cerebrovascular events. To test this, we investigated sex differences in the prevalence of MRI detectable plaque hemorrhage (MRI PH) as an index of plaque instability, and secondly whether MRI PH mediates sex differences in the rate of cerebrovascular recurrence.

Methods

Prevalence of PH between sexes was analysed in a single centre pooled cohort of 176 patients with recently symptomatic, significant carotid stenosis (106 severe [≥70%], 70 moderate [50–69%]) who underwent prospective carotid MRI scanning for identification of MRI PH. Further, a meta-analysis of published evidence was undertaken. Recurrent events were noted during clinical follow up for survival analysis.

Results

Women with symptomatic carotid stenosis (50%≥) were less likely to have plaque hemorrhage (PH) than men (46% vs. 70%) with an adjusted OR of 0.23 [95% CI 0.10–0.50, P<0.0001] controlling for other known vascular risk factors. This negative association was only significant for the severe stenosis subgroup (adjusted OR 0.18, 95% CI 0.067–0.50) not the moderate degree stenosis. Female sex in this subgroup also predicted a longer time to recurrent cerebral ischemic events (HR 0.38 95% CI 0.15–0.98, P = 0.045). Further addition of MRI PH or smoking abolished the sex effects with only MRI PH exerting a direct effect.Meta-analysis confirmed a protective effect of female sex on development of PH: unadjusted OR for presence of PH = 0.54 (95% CI 0.45–0.67, p<0.00001).

Conclusions

MRI PH is significantly less prevalent in women. Women with MRI PH and severe stenosis have a similar risk as men for recurrent cerebrovascular events. MRI PH thus allows overcoming the sex bias in selection for CEA.     

Structural, biochemical, and in vivo characterization of the first virally encoded cyclophilin from the Mimivirus

Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses.