1,720,995 research outputs found
Plant lipid signaling protocols
No full textEukaryotic cells are surrounded by membranes consisting of various lipids, including sterols, sphingolipids, glycolipids, and phospholipids. Besides structural functions, membranes also contain lipids with regulatory and signaling roles. Such lipids include polyphosphoinositides, the low-abundant derivatives of phosphatidylinositol, phosphatidic acid, certain sphingolipids, N -acyl ethanolamines, and oxylipins, oxidized derivatives of C18-fatty acids; all these are emerging as important players regulating various physiological processes in plants. As we are beginning to understand the complexity of lipid signaling and its roles in plant biology, there is an increasing interest in their analysis. However, due to the low abundance and transient nature of some of these hydrophobic compounds, this is not always easy. The dif fi culty in analyzing signaling lipids arises from some unique analytical challenges. For instance, the structural similarity of polyphosphoinositide species and of derived soluble inositol polyphosphates must be taken into account when using "speci fi c" in vivo markers or when assaying relevant enzyme activities. Furthermore, plants exhibit greater metabolic complexity in comparison to animals or yeast, and the detection of low levels of signaling molecules requires specialized technology. Together, these considerations have created a need for novel means to identify and quantify signaling lipids and have raised new questions regarding the biochemical properties of enzymes involved in their conversion. This book is dedicated to the various experimental approaches by which plant signaling lipids can be studied. Experts in the fi eld have contributed their knowledge and extensive experience to provide a collection of 27 chapters on the analysis of plant signaling lipids, including detailed protocols to detect various relevant compounds by targeted or nontargeted approaches; to assay relevant enzyme activities in biological material or using recombinant enzymes; to test for speci fi c binding of signaling lipids to protein partners; or to visualize signaling lipids or lipid-derived signals in living plant cells. On the way, a broad spectrum of methods is detailed, such as radiolabeling, diverse chromatographic methods (TLC, HPLC, GC), mass spectrometry, nuclear magnetic resonance spectroscopy, and confocal imaging. By looking left and right at signaling events related to lipids, key protocols for the analysis of other important players are also covered, such as diacylglycerol, galactolipids, and Ca 2+ . All methods described have been developed or optimized in particular for the use in plants and are described including proper controls and notes for meaningful interpretation to ensure that tools and assays are used correctly and to their full potential. Tools presented often provide alternative options for relevant analyses, either requiring extensive instrumentation or being achievable with the most basic of laboratory equipment, thus helping interested researchers to select methods suitable for their questions and equipment available. Research groups around the world are pursuing an increasing number of questions about functions of plant lipid signals, including adaptation to environmental stresses, guard cell functioning, vesicle traf fi cking, or control in development, such as cell polarity and pattern formation. Paying tribute to its emerging complexity, we are convinced that plant lipid signaling is best approached by multiple parallel methods, combining key biochemical tests and cell biological analyses described in this book. It is therefore timely and important to present plant researchers with this full set of tools needed to elucidate the particular roles of signaling lipids in plants. We hope that this collection of current protocols will aid plant researchers in their endeavor to elucidate the roles of lipid signals, and we are eager to see these methods put to use towards exciting new discoveries
Analysis of D3-,4-,5-phosphorylated phosphoinositides using HPLC
No full textDetection of polyphosphoinositides (PPIs) is difficult due to their low chemical abundancy. This problem is further complicated by the fact that PPIs are present as various, distinct isomers, which are difficult, if not impossible, to separate by conventional thin layer chromatography (TLC) systems. PPIs in plants include PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P 2, and PtdIns(4,5)P 2. Here, a protocol is described analyzing plant PPIs using (32)P-orthophosphorus pre-labeled material. After extraction, lipids are deacylated and the resulting glycerophosphoinositol polyphosphates (GroPInsPs) separated by HPLC using a strong anion-exchange column and a shallow salt gradient. Alternatively, PPIs are first separated by TLC, the lipids reisolated, deacylated, and the GroPInsPs then separated by HPLC
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Get PDFWe 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
Analyzing Plant Signaling Phospholipids Through 32Pi-Labeling and TLC
No full textLipidomic analyses through LC-, GC-, and ESI-MS/MS can detect numerous lipid species based on headgroup and fatty acid compositions but usually miss the minor phospholipids involved in cell signaling because of their low chemical abundancy. Due to their high turnover, these signaling lipids are, however, readily picked up by labeling plant material with (32)P-orthophosphate and subsequent analysis of the lipid extracts by thin layer chromatography. Here, protocols are described for suspension-cultured tobacco BY-2 cells, young Arabidopsis seedlings, Vicia faba roots, and Arabidopsis leaf disks, which can easily be modified for other plant species and tissues
Lipid-binding analysis using a fat blot assay
No full textProtein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose membrane-immobilized lipids. The assay is adapted from the method by Dowler et al. (Sci STKE 129:pl6, 2002) and provides qualitative and quantitative information on the relative affinity with which a protein binds to a particular lipid. To perform a Fat Blot assay, serial dilutions of different phospholipids are spotted onto a nitrocellulose membrane. These membranes are then incubated with a lipid-binding protein possessing a GST (or other epitope) tag. The membranes are washed and the protein, which is bound to the membrane by virtue of its interaction with the lipid's head group, is detected by immunoblotting with an antibody against GST (or other epitope). The procedure only requires a few micrograms of protein and is quick, simple and cheap to perform
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
- …
