An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength

A simple, rapid, and inexpensive method for the preparation and purification of chloroplast DNA (cpDNA) from pea has been developed. The crucial step is the isolation of chloroplasts in a medium of high ionic strength (I congruent equal to 1.40 M). CpDNA from pea prepared according to this method has successfully been used for restriction enzyme mapping, Southern transfers, and cloning.     

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated froms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on μBondapak C₁₈ coupled with uv detection allows direct quantitation of the folates in the nanogram range.     

Anwendung der automatisierten Festphasenextraktion bei der Bestimmung von Ochratoxin A

For some foodstuffs, determination of the mycotoxin ochratoxin A (OTA) requires time consuming clean up by means of solid phase extraction (SPE). Therefore a system for automated SPE was tested for cleaning up roasted coffee as a possible way of shortening preparation time. Validation of the method in accordance to the so called “Concept '98” led to a LOD of 0.2 μg/kg and a recovery rate of 92%. By using the described procedure with samples of roasted coffee the OTA contents varied between the LOD and 3.4 μg/kg. This method was also used to determine ochratoxin A in liquorice roots, ginger and valerian.

Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004     

Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development

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An overview of mechanistic modeling of liquid chromatography

Abstract

Liquid chromatography is considered to be the bottleneck for purification of therapeutic proteins. Development and optimization of chromatography process is a cumbersome activity due to the increasing complexities in the types and content of impurities present in the high product titer cell culture harvest obtained from the upstream processing. Further, regulatory expectations are continuously rising with the recent initiatives of quality by design and process analytical technology expecting the manufacturer to have a deeper understanding of the process and the product. Mechanistic modeling is one approach to gain this deeper understanding of a process step. It involves modeling of the underlying physicochemical processes. A well calibrated model with acceptable predictability can be very effective in both process optimization and process characterization activities. In this paper we provide an overview of mechanistic modeling of liquid chromatography. We discuss the various components that such a model entails and also presents the status quo of this area.     

The role of the lipid matrix in the biosynthesis of dolichyl-linked oligosaccharides

The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol; DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol This revised version was published online in November 2006 with corrections to the Cover Date.