Role of the B1 short arm in laminin self-assembly

Laminin self-assembles into a basement membrane polymer through specific low-affinity interactions. Recently, it was shown that the terminal short-arm domain (domains VI and V) of the B1 chain (fragment E4) possesses one of the laminin self-interaction sites [Schittny, J.C. & Yurchenco, P.D. (1990) J. Cell Biol. 110, 825-832], but that the binding partner(s) of this domain is unknown. Using affinity retardation chromatography we now investigate the domain(s) fragment E4 binds to. The elution of E4 was clearly retarded on immobilized laminin and fragment E1' (three-chain short-arm complex excluding the distal part of the B1 chain), but not on immobilized E4 in calcium containing buffer and at 37 degrees C. Under the same conditions, E1' strongly interacts with immobilized E4. In addition, E1' is able to non-covalently cross-link soluble E4 to immobilized E4. No further interaction of laminin and E4 with additional fragments (P1', A, B2 and B1 chain short-arm complex without B1-domains VI-IV and without globules; E8, distal long arm and G1-3; E3, long-arm G subdomains 4 and 5) could be demonstrated. These data are interpreted as evidence that (a) the primary laminin-laminin bonds are formed between the short arms of laminin, that (b) the terminal B1 short-arm domain (E4) can interact with the short arm(s) of the A and/or B2 chain(s) (domain E1'), but does not self-interact, and that (c) due to at least three self-binding sites, laminin polymerization behaves co-operatively.

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.

Личните и географските имена в българския и сръбския превод на Стишния пролог (по материал от месец март)

The paper presents a comparative study of the personal and geographic names in the two complete Slavic translations of the Byzantine Versified Synaxarion, namely the Bulgarian and Serbian Prolog Stišnoj, which appeared around the first half of the 14 th century. On the basis of the March texts in seven South Slavic manuscripts, the differences in the rendering of the personal names are analysed on the level of phonetics, orthography, morphology and word formation. The data allow the following conclusions: 1) The differences in the forms of these names in the Bulgarian and Serbian Prolog Stišnoj give further arguments supporting their independent origin; 2) Several specific tendencies are noted which more or less differentiate them. The Bulgarian translation reproduces more accurately the graphics of the original names, allows dativus possessivus, often replaces the Greek anthroponyms and toponyms with adjectives, presents many local names in the plural, and sometimes retains Greek nominative endings in masculine personal names. The Serbian translation, on the other hand, follows more often the Byzantine pronunciation of the names, complies more strictly with their grammatical characteristics (case, number), separates more often the ending -ς from the stem, and incorporates the accusative ending -ν into the stem of certain anthroponyms several times. 3) The tendency towards Slavicisation of the personal names is nearly the same in both translations and cannot be viewed as peculiar of either of them.

Molecular epidemiology of Bacillus anthracis: determining the correct origin

We analyzed and compared strains of Bacillus anthracis isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human B. anthracis strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurred between 1978 and 1981 in a factory processing cashmere wool from the Indian subcontinent. We interpret the worldwide occurrence of B. anthracis strains of cluster A4 to be due to the extensive global trade of untreated cashmere wool during the last century.

Use of dextran sulfate in tourniquet-induced skeletal muscle reperfusion injury.

BACKGROUND Lower extremity ischemia-reperfusion injury (IRI)-prolonged ischemia and the subsequent restoration of circulation-may result from thrombotic occlusion, embolism, trauma, or tourniquet application in surgery. The aim of this study was to assess the effect of low-molecular-weight dextran sulfate (DXS) on skeletal muscle IRI. METHODS Rats were subjected to 3 h of ischemia and 2 or 24 h of reperfusion. To induce ischemia the femoral artery was clamped and a tourniquet placed under the maintenance of the venous return. DXS was injected systemically 10 min before reperfusion. Muscle and lung tissue samples were analyzed for deposition of immunoglobulin M (IgM), IgG, C1q, C3b/c, fibrin, and expression of vascular endothelial-cadherin and bradykinin receptors b1 and b2. RESULTS Antibody deposition in reperfused legs was reduced by DXS after 2 h (P < 0.001, IgM and IgG) and 24 h (P < 0.001, IgM), C3b/c deposition was reduced in muscle and lung tissue (P < 0.001), whereas C1q deposition was reduced only in muscle (P < 0.05). DXS reduced fibrin deposits in contralateral legs after 24 h of reperfusion but did not reduce edema in muscle and lung tissue or improve muscle viability. Bradykinin receptor b1 and vascular endothelial-cadherin expression were increased in lung tissue after 24 h of reperfusion in DXS-treated and non-treated rats but bradykinin receptor b2 was not affected by IRI. CONCLUSIONS In contrast to studies in myocardial infarction, DXS did not reduce IRI in this model. Neither edema formation nor viability was improved, whereas deposition of complement and coagulation components was significantly reduced. Our data suggest that skeletal muscle IRI may not be caused by the complement or coagulation alone, but the kinin system may play an important role.

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118.

Interactions between Eph receptors and their membrane-bound ligands (ephrins) are of critical importance for key developmental processes such as boundary formation or vascular development. Their downstream signaling pathways are intricate and heterogeneous at several levels, the combined effect being a highly complex and flexible system. Here we demonstrate that activated EphB1 induces tyrosine phosphorylation of the focal adhesion protein paxillin at Tyr-31 and Tyr-118 and is recruited to paxillin-focal adhesion kinase (FAK) complexes. Pretreatment with the specific Src inhibitor PP2, or expression of dominant-negative, kinase-dead c-Src abrogates EphB1-induced tyrosine phosphorylation of paxillin. Cells transfected with the paxillin mutant Y31F/Y118F displayed a reduced migration in response to ephrin B2 stimulation. Furthermore, expression of an LD4 deletion mutant (paxillin DeltaLD4) significantly reduces EphB1-paxillin association, paxillin tyrosine phosphorylation, as well as EphB1-dependent cell migration. Finally, mutation of the Nck-binding site of EphB1 (Y594F) interrupts the interaction between Nck, paxillin, and EphB1. These data suggest a model in which ligand-activated EphB1 forms a signaling complex with Nck, paxillin, and focal adhesion kinase and induces tyrosine phosphorylation of paxillin in a c-Src-dependent manner to promote cell migration.

Benchmark problems for reactive transport modeling of the generation and attenuation of acid rock drainage

Acid rock drainage (ARD) is a problem of international relevance with substantial environmental and economic implications. Reactive transport modeling has proven a powerful tool for the process-based assessment of metal release and attenuation at ARD sites. Although a variety of models has been used to investigate ARD, a systematic model intercomparison has not been conducted to date. This contribution presents such a model intercomparison involving three synthetic benchmark problems designed to evaluate model results for the most relevant processes at ARD sites. The first benchmark (ARD-B1) focuses on the oxidation of sulfide minerals in an unsaturated tailing impoundment, affected by the ingress of atmospheric oxygen. ARD-B2 extends the first problem to include pH buffering by primary mineral dissolution and secondary mineral precipitation. The third problem (ARD-B3) in addition considers the kinetic and pH-dependent dissolution of silicate minerals under low pH conditions. The set of benchmarks was solved by four reactive transport codes, namely CrunchFlow, Flotran, HP1, and MIN3P. The results comparison focused on spatial profiles of dissolved concentrations, pH and pE, pore gas composition, and mineral assemblages. In addition, results of transient profiles for selected elements and cumulative mass loadings were considered in the intercomparison. Despite substantial differences in model formulations, very good agreement was obtained between the various codes. Residual deviations between the results are analyzed and discussed in terms of their implications for capturing system evolution and long-term mass loading predictions.

Worksite Tobacco Prevention: A Randomized, Controlled Trial of Adoption, Dissemination Strategies, and Aggregated Health-Related Outcomes across Companies

Evidence based public health requires knowledge about successful dissemination of public health measures. This study analyses (a) the changes in worksite tobacco prevention (TP) in the Canton of Zurich, Switzerland, between 2007 and 2009; (b1) the results of a multistep versus a “brochure only” dissemination strategy; (b2) the results of a monothematic versus a comprehensive dissemination strategy that aim to get companies to adopt TP measures; and (c) whether worksite TP is associated with health- related outcomes. A longitudinal design with randomized control groups was applied. Data on worksite TP and health-related outcomes were gathered by a written questionnaire (baseline