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.     

CD38 and CD157 as receptors of the immune system: a bridge between innate and adaptive immunity

This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage.     

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.     

Analysis of the Influences of Short-Term Levosimendan Exposure on Oxidant/Antioxidant Status and Trace-Element Levels in the Physiological Status of the Thoracic Aorta of Rats

The objective of this study was to evaluate the effect of levosimendan (chemical formula C₁₄H₁₂N₆O) exposure on oxidant/antioxidant status and trace-element levels in the thoracic aorta of rats. Eighteen male Wistar albino rats were randomly divided into two groups of eight animals each. Group 1 was not exposed to levosimendan and served as a control. Levosimendan (12???g/kg) diluted in 10 ml 0.5?% dextrose was administered intraperitoneally to group 2. Animals of both groups were killed after 3?days, and their thoracic aortae were harvested for determination of changes in tissue oxidant/antioxidant status and trace-element levels. The animals in both groups were killed 72?h after levosimendan exposure, and thoracic aortae were harvested for determination of the lipid peroxidation product MDA and antioxidant GSH levels and the activities of antioxidant enzymes such as SOD, GSH-Px and CAT. It was found that MDA, GSH and CAT enzyme levels increased in thoracic aortae of rats after levosimendan administration. SOD and CA enzyme activities and the level of antioxidant GSH decreased in thoracic aortae of rats after levosimendan treatment. Pb, Cd and Fe levels of thoracic aortae were significantly higher (P?<?0.001) and Mg, Mn, Zn and Cu were significantly lower (P?<?0.001) in the levosimendan group compared to the control group. These results suggest that short-term levosimendan treatment caused an increase in free radical production and a decrease in antioxidant enzyme activity in thoracic aortae of levosimendan-treated rats. It also causes a decrease or increase in many mineral levels of the thoracic aorta, which is an undesirable condition for normal pharmacological function.     

The bioactive peptides salusins and apelin-36 are produced in human arterial and venous tissues and the changes of their levels during cardiopulmonary bypass

This study aimed to examine the effects of CPB on salusin-α, salusin-β and apelin-36 bioactive peptides in people who are planned to undergo coronary artery bypass graft (CABG) operation due to coronary artery disease and to explore whether these peptides are produced in human aortic, saphenous and arterial tissues. The study included age and BMI matched 15 patients who underwent CABG operation by CPB. In order to determine salusin-α, salusin-β and apelin-36 levels, venous blood samples were collected before induction of anesthesia (T1), before CPB (T2), 5min before the removal of cross-clamp (T3), 5min after the removal of cross-clamp (T4), upon arrival in the intensive care (T5), at postoperative 24th hour (T6) and 72nd hour (T7). Salusin and apelin expressions of the tissues were shown by immunohistochemical method. Peptide amounts of sera and tissues were measured using ELISA. Salusins production by vessels occurs in fibroblast cells of the media in the aorta and smooth muscle cells of the media in the LIMA and saphena. Apelin is produced by endothelial cells of the intima and fibroblast cells of the media in the aorta and by smooth muscle cells of the media in the LIMA and saphena. Changes in the levels of salusin-β and apelin-36 were significant during CPB. Salusin-α, salusin-β and apelin-36 are locally synthesized in the arteries and veins. Salusins and apelin-36 might be important markers in the CPB, and also that salusin-β was more specific in comparison to salusin-α.