Membrane-induced folding and structure of membrane-bound annexin A1 N-terminal peptides: implications for annexin-induced membrane aggregation

Annexins constitute a family of calcium-dependent membrane-binding proteins and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an α-helical conformation and has been implicated in mediating the membrane aggregation behavior of this protein. Although the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane-binding site of annexin A1. This study used circular dichroism spectroscopy to show that a rat annexin A1 N-terminal peptide possesses random coil structure in aqueous buffer but an α-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveals an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane. Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into α-helical structures and that this conformation enables favorable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor.     

Esp1396I restriction-modification system: structural organization and mode of regulation

Esp1396I restriction–modification (RM) system recognizes an interrupted palindromic DNA sequ ence 5′-CCA(N)₅TGG-3′. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.     

Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition

Src kinase controls cellular adhesions, including cadherin-based intercellular adhesions and integrin-mediated cell-matrix adhesions. In epithelial cells, Src activation, or increased signalling from migratory growth factor receptors via Src, induces an adhesion switch that enhances dynamic cell-matrix adhesions and migratory capacity while suppressing intercellular contact. Moreover, Src and the associated tyrosine kinase FAK are at the heart of the recently identified crosstalk between integrin- and cadherin-mediated adhesions of epithelial cells, particularly during the epithelial-to-mesenchymal transition.     

Solubility of multi-component biodiesel fuel systems

Solubility of biodiesel fuel components in fossil diesel fuel-methanol-rapeseed oil methyl ester, fossil diesel fuel-ethanol-rapeseed oil methyl ester and fossil diesel fuel-ethanol-rapeseed oil ethyl ester systems was investigated. The solubility of components in the fossil diesel fuel-ethanol-rapeseed oil methyl ester system at 20 degrees C was substantially higher than in the fossil diesel fuel-methanol-rapeseed oil methyl ester system. The solubility of components in the fossil diesel fuel-ethanol-rapeseed oil ethyl ester system was slightly lower than in the fossil diesel fuel-ethanol-rapeseed oil methyl ester mixture. The moisture content of ethanol had a great influence on mixture solubility. With decrease of temperature, the solubility of components in the fossil diesel fuel-ethanol-rapeseed oil methyl ester system decreased.     

The timing and extent of activation of diacylglycerol-responsive protein kinase-cs determines their ability to inhibit or promote platelet-derived growth factor-dependent DNA synthesis

In response to natural agonists, such as platelet-derived growth factor (PDGF), diacylglycerol-responsive protein kinase Cs (PKCs) are activated at two distinct times, early and mid G1, and only the late activity is required for the transition into S phase. Surprisingly, the potent PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits DNA synthesis when it is added in mid G1. Here we investigated why different PKC agonists had opposing effects on cell proliferation. We found that the magnitude and timing of PKC activation determined their ability to suppress DNA synthesis. Furthermore, potent activation of PKCs resulted in robust Erk activation and elevation of p21(CIP1). Finally, PMA was unable to block PDGF-dependent cell cycle progression in cells that lack p21(CIP1). These findings indicate that only potent activators of PKC were capable of blocking cell cycle progression, and the mechanism appears to involve an elevation of p21(CIP1).     

Annexin 1 modulates monocyte-endothelial cell interaction in vitro and cell migration in vivo in the human SCID mouse transplantation model

The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; approximately 50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1alpha (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo.     

Über den Einfluß niedriger und hoher O2-Partialdrucke auf den Sauerstoff- und Kohlendioxidumsatz von Amaranthus und Phaseolus während der Lichtphase

Summary Carbon dioxide and oxygen gas exchange of illuminated Amaranthus and Phaseolus leaves was measured from 0–600 ppm of CO₂ in an open system.At low oxygen concentration (2% O₂) the ratio of CO₂ uptake to O₂ evolution came close to 1.At high oxygen partial pressure (42% O₂) the O₂ compensation point of an Amaranthus leaf was increased and oxygen evolution was depressed. Accordingly the CO₂/O₂ quotients were variable; the lowest value of 1,9 differed significantly from 1,0.The oxygen and carbon dioxide compensation points of a Phaseolus leaf were increased at high oxygen concentration (42% O₂) and oxygen evolution as well as carbon dioxide uptake were reduced. Therefore the ratios CO₂ over O₂ varied and differed greatly from 1,0.It was concluded that the nature of photosynthates is regulated by the gas composition around the leaves.     

Increased nest predation near protected capercaillie leks: a caveat against small reserves

Protecting animal aggregation sites is intuitively an efficient conservation approach, particularly for threatened species in fragmented landscapes. However, the appropriate scale of protection depends on accompanying threats, such as predator attraction to the same aggregation sites, to which the target species may need to respond by moving to alternative habitat patches. We performed experiments using artificial ground nests around protected capercaillie leks in commercially managed forest landscape in Estonia. We considered two scales: the landscape up to 3 km from the lek centre, and among 10–30-ha forest compartments, which differed in predator abundance. We found that nest predation significantly declined with distance from the lek. Nests were depredated by multiple mammalian and avian predator species and total predator abundance explained most of the between-forest variation in predation rate. Our results indicate that, in this hemiboreal area, (i) ground nest predation is largely determined by landscape-scale distribution of predators and (ii) predators can aggregate at capercaillie leks. The implication is that lek-centred habitat protection used for the capercaillie and other grouse may be ineffective unless the peripheral habitat quality and predator abundance are specifically addressed. More generally, predation pressure can be a serious problem in small set-asides within hostile landscapes for threatened ground-nesting birds.     

Acetylcholine inhibits cell cycle progression in rat Schwann cells by activation of the M2 receptor subtype

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.     

Fast, fully automated analysis of voriconazole from serum by LC-LC-ESI-MS-MS with parallel column-switching technique

Voriconazole is a novel broad-spectrum antifungal agent. We developed an on-line LC-LC-MS-MS method for fully automated and direct analysis of voriconazole in raw human serum. After injection of human serum size-selective sample fractionation and analyte extraction was achieved using an extraction column (25 mm x 4 mm) packed with a restricted access material (RAM, LiChrospher ADS C(8), 25 microm). On-line transfer of voriconazole from the extraction column was followed by chromatography separation on a C(18) column. Detection was done by ESI-MS-MS. The total analysis time was 13 min, managed by parallel extraction and chromatographic separation. This LC-MS assay was fully validated. The lower limit of quantification was 0.05 microg/ml. The automated inline extraction of voriconazole described here eliminates the need for difficult and time-consuming sample pre-treatment. Other advantages of the new method are that only a small quantity (5 microl) of serum is needed and that the method is very specific.