1,720,977 research outputs found
A practical guide to insect cell cultures: establishment and maintenance of primary cell cultures
Full text linkIn the last three decades several insect species have been studied in vitro using cell culture revealing fundamental information about their biology. However, numerous very important species have never been investigated at a cellular level so that insect cell culture is still a field in an early stage of its potential development. In the present review, we summarized the main steps involved in the establishment of primary cell cultures to serve as a practical guide for current and future entomologists to leverage the power of cell cultures. This approach has the potential to generate valuable results and suggestions about insect metabolism, vectorial capacity and adaptation to different stresses and challenges. Although most published papers discussed immortalized cell lines, we focussed our review on primary cell culture since they can give precise data in different research fields
THE NATURAL ARG386CYS MUTATION IN THE COAGULATION FACTOR X, AS A TOOL TO INVESTIGATE DIFFERENT FX-REQUIREMENTS IN INITIATION AND PROPAGATION PHASES.
No full textBackground: The natural coagulation factor X (FX) substitution FXR386C, affecting the catalytic domain, provided us with a tool to investigate TF/FVIIa and FVIIIa/FIXa -dependent FX activation.
Materials and methods: The natural (R386C) and designed (R386A and the inactive S379A-R386C) FX mutants were stably expressed in Human Embrionic Kidney cells and purified by ion exchange, immunoaffinity and hydroxyapatite chromatography.
In vitro secretion and in vivo stability in mice of recombinant molecules were studied. Variants were characterized by coagulation tests (PT and APTT) and functional assays in plasma and in reconstituted systems.
Results: Alignment of mammalian sequences of vitamin K-dependent coagulation factors showed that the R386 residue is not conserved among serine-proteases. Moreover, in the crystallographic structure of FXa it is exposed on the surface of the catalytic domain.
Major alteration of FX biosynthesis or function of the 386C variant were excluded by its normal i) secretion in vitro (1008±504 ng/ml vs 813±190 ng/ml of rFXwt), ii) clearance in vivo iii) procoagulant activity in a PT-based assay (rFX386C 105,7±5,7%). PT activity in plasma of the rFX386A and rFX379A386C variants were 173.6±7.1% and 0±0%, respectively. Instead, significantly reduced APTT values (rFX386C 28.7±0.5%, rFX386A 52.3±0.6%, rFX379A386C 0±0%) suggested impaired intrinsic activation of mutants with substitution at the 386 position.
Functional assays exploiting fluoro/chromogenic substrates were used to dissect activation and activity of FX variants. Extrinsic activation, amydolytic and thrombin generation activities were indistinguishable from those of rFXwt. Noteworthy, a significant variation in kinetic parameters was measured for the rFX386C and rFX386A mutants in FXa generation assay upon activation by purified FVIIIa/FIXa complex.
Conclusions: The A386C substitution in the coagulation FX is compatible with normal protein biosynthesis and almost exclusively affects its intrinsic activation. The 386 residue is potentially included in a functional exosite involved in macromolecular interactions
Biochemical characterization of the natural Cys386-FX variant: a new tool to investigate coagulation factor X functions?
No full text
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Characterization of the intracellular signalling capacity of natural FXa mutants with reduced pro-coagulant activity
No full textIntroduction: Factor X (FX) is a serine-protease playing a crucial
role in the blood coagulation pathway and triggering intracellular signalling in
a variety of cells via protease-activated receptors (PARs). By exploiting
naturally occurring variants (V342A and G381D, catalytic domain; E19A, γ-
carboxyglutamic acid (GLA)-rich domain), we investigated the relationship
between the pro-coagulant activity and the signal transduction capacity of FX.
Materials and methods: Recombinant FX (rFX) variants were expressed in Human
Embryonic Kidney cells and purified by immunoaffinity chromatography. Activated
rFX (rFXa) variants were characterized for pro-coagulant, amidolytic and
thrombin generation activity. rFXa signalling was assessed through evaluation of
extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in C2C12
myoblasts.
Results and conclusions: rFX variants showed reduced (rFX-342A, 29%; rFX-19A,
12%) or not detectable (rFX-381D) amidolytic activity. Thrombin generation
activity in a plasma system was also decreased either upon activation by
Russell's viper venom (rFX-342A, 38%; rFX-19A, 7%; rFX-381D, not detectable) or
by the extrinsic pathway (rFX-342A, 36%; rFX-19A, rFX-381D, not detectable). The
rFXa-381D mutant displayed little or no enzymatic activity, and did not induce
any appreciable signal transduction capacity. The rFXa-342A mutant induced a
dose-dependent signalling with a 50% reduced signalling capacity. At the highest
concentration (174 nM), signalling progressed with a time course similar to that
of rFXa-wt. Zymogen rFX-19A showed defective and incomplete activation resulting
in strongly reduced enzymatic activity and signalling. Taken together our data
are consistent with a close correlation between pro-coagulant activity and
intracellular signalling capacity
- …
