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[
1987]
Current knowledge of sterol biochemistry and physiology in nematodes is reviewed. Nematodes possess a nutritional requirement for sterol because they lack the capacity for de novo sterol biosynthesis. The free-living nematode Caenorhabditis elegans has recently been used as a model organism for investigation of nematode sterol metabolism. C. elegans is capable of removal of the C-24 alkyl substituent of plant sterols such as sitosterol and also possesses the remarkable ability to attach a methyl group at C-4 on the sterol nucleus. An azasteroid and several long-chain alkyl amines disrupt the phytosterol dealkylation pathway in C. elegans by inhibiting its *24-sterol reductase. These compounds inhibit growth and reproduction in certain parasitic nematodes and provide model compounds for development of novel nematode control
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[
Methods Mol Biol,
2011]
Quantitative proteomics aims to identify and quantify proteins in cells or organisms that have been obtained from different biological origin (e.g., "healthy vs. diseased"), that have received different treatments, or that have different genetic backgrounds. Protein expression levels can be quantified by labeling proteins with stable isotopes, followed by mass spectrometric analysis. Stable isotopes can be introduced in vitro by reacting proteins or peptides with isotope-coded reagents (e.g., iTRAQ, reductive methylation). A preferred way, however, is the metabolic incorporation of heavy isotopes into cells or organisms by providing the label, in the form of amino acids (such as in SILAC) or salts, in the growth media. The advantage of in vivo labeling is that it does not suffer from side reactions or incomplete labeling that might occur in chemical derivatization. In addition, metabolic labeling occurs at the earliest possible moment in the sample preparation process, thereby minimizing the error in quantitation. Labeling with the heavy stable isotope of nitrogen (i.e., (15)N) provides an efficient way for accurate protein quantitation. Where the application of SILAC is mostly restricted to cell culture, (15)N labeling can be used for micro-organisms as well as a number of higher (multicellular) organisms. The most prominent examples of the latter are Caenorhabditis elegans and Drosophila (fruit fly), two important model organisms for a range of regulatory processes underlying developmental biology. Here we describe in detail the labeling with (15)N atoms, with a particular focus on fruit flies and C. elegans. We also describe methods for the identification and quantitation of (15)N-labeled proteins by mass spectrometry and bioinformatic analysis.
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[
1989]
Classical embryological studies of nematodes, primarily by Van Beneden and Boveri near the turn of the century, have made lasting contributions to our understanding of embryonic development (1). However, during most of this century, nematodes have been eclipsed as a model system for embryology by organisms with more tractable embryos such as sea urchins, insects, amphibians, birds, and mice. Two features of the free-living soil nematode Caenorhabditis elegans have returned nematodes to a prominent place in embryological investigations: its suitability for genetic analysis and its invariant and completely described cell lineage. These two features, combined with technological advances in microscopy and molecular biology, are providing the opportunity to combine experimental embryology with genetic and molecular analyses of embryonic development at the level of individual cells in a single organism. This chapter focuses on efforts to understand the molecular and cellular events of early development in C. elegans with particular emphasis on events relating to the determination of embryonic cell fates. Extensive coverage of the various contributions that the study of Caenorhabditis has made to our knowledge of developmental biology can be found in ref. 2.
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[
Methods Cell Biol,
2012]
In Caenorhabdatis elegans as in other animals, fat regulation reflects the outcome of behavioral, physiological, and metabolic processes. The amenability of C. elegans to experimentation has led to utilization of this organism for elucidating the complex homeostatic mechanisms that underlie energy balance in intact organisms. The optical advantages of C. elegans further offer the possibility of studying cell biological mechanisms of fat uptake, transport, storage, and utilization, perhaps in real time. Here, we discuss the rationale as well as advantages and potential pitfalls of methods used thus far to study metabolism and fat regulation, specifically triglyceride metabolism, in C. elegans. We provide detailed methods for visualization of fat depots in fixed animals using histochemical stains and in live animals by vital dyes. Protocols are provided and discussed for chloroform-based extraction of total lipids from C. elegans homogenates used to assess total triglyceride or phospholipid content by methods such as thin-layer chromatography or used to obtain fatty acid profiles by methods such as gas chromatography/mass spectrometry. Additionally, protocols are provided for the determination of rates of intestinal fatty acid uptake and fatty acid breakdown by -oxidation. Finally, we discuss methods for determining rates of de novo fat synthesis and Raman scattering approaches that have recently been employed to investigate C. elegans lipids without reliance on invasive techniques. As the C. elegans fat field is relatively new, we anticipate that the indicated methods will likely be improved upon and expanded as additional researchers enter this field.