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Proteomic analysis of Caenorhabditis elegans.
Jeong, Pan-Young,Na, Keun,Jeong, Mi-Jeong,Chitwood, David,Shim, Yhong-Hee,Paik, Young-Ki Humana Press 2009 METHODS IN MOLECULAR BIOLOGY -CLIFTON THEN TOTOWA- Vol.519 No.-
<P>Proteomic studies of the free-living nematode Caenorhabditis elegans have recently received great attention because this animal model is a useful platform for the in vivo study of various biological problems relevant to human disease. In general, proteomic analysis is carried out in order to address a specific question with respect to differential changes in proteome expression under certain perturbed conditions. In this chapter, we focus on gel-based proteomic analysis of C. elegans subjected to two specific stress conditions during development: induction of the dauer state for whole body protein expression and a temperature shift for egg protein expression. Utilizing these differently perturbed C. elegans protein samples, two-dimensional electrophoresis and differential in-gel electrophoresis methods have led to the discovery of remarkable aspects of the worm's biology. We also provide numerous details about the technical points and protocols necessary for successful experimentation.</P>
Effects of sterols on the development and aging of Caenorhabditis elegans.
Lee, Eun-Young,Jeong, Pan-Young,Kim, Sun-Young,Shim, Yhong-Hee,Chitwood, David J,Paik, Young-Ki Humana Press 2009 Methods in molecular biology Vol.462 No.-
<P>Although Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterols as essential nutrients. Supplemental cholesterol undergoes extensive enzymatic modification in C. elegans to form certain sterols of unknown function. Since sterol metabolism in C. elegans differs from that in other species, such as mammals and yeast, it is important to examine how sterols regulate worm physiology. To examine the functions of sterols in C. elegans, a sterol-feeding experiment was carried out and several critical parameters, such as brood size, growth rate, and life span, were measured. In addition, the change in lipid distribution in C. elegans can be both qualitatively and quantitatively determined by various methods, including staining and chromatographic techniques. Taken together, the effects of sterols on C. elegans are very prominent and can be easily assessed using the techniques described here.</P>
Proteomic analysis of the sterol-mediated signaling pathway in Caenorhabditis elegans.
Choi, Byung-Kwon,Shin, Yun-Kyung,Lee, Eun-Young,Jeong, Pan-Young,Shim, Yhong-Hee,Chitwood, David J,Paik, Young-Ki Humana Press 2009 Methods in molecular biology Vol.462 No.-
<P>Since Caenorhabditis elegans is incapable of de novo cholesterol biosynthesis, it must utilize other nonpermissive sterols that are present in the environment by converting them into cholesterol for cellular function. The inhibition of sterol conversion to cholesterol in C. elegans by various sterol biosynthesis inhibitors (SBIs) is known to cause serious defects in the development of these worms. To determine the biochemical consequences of these physiological abnormalities, one can perform a proteomic analysis of worms of a certain stage that are grown in the presence of SBIs in order for the differential expression of proteins involved in the sterol-mediated signaling pathway to be identified. For example, reductions in the expression of lipoprotein family members, such as vitellogenin-2 and vitellogenin-6, are prominent in azacoprostane-treated worms. This phenomenon is also seen in worms treated with AY-9944, which blocks the conversion of 7-dehydrocholesterol, a major sterol present in C. elegans, to cholesterol.</P>
Joo, Hyoe-Jin,Yim, Yong-Hyeon,Jeong, Pan-Young,Jin, You-Xun,Lee, Jeong-Eui,Kim, Heekyeong,Jeong, Seul-Ki,Chitwood, David J,Paik, Young-Ki Biochemical Society 2009 The Biochemical journal Vol.422 No.1
<P>Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.</P>
Lee, Joon-Hee,Choi, Sun-Hye,Kwon, Oh-Seung,Shin, Tae-Joon,Lee, Jun-Ho,Lee, Byung-Hwan,Yoon, In-Soo,Pyo, Mi Kyung,Rhim, Hyewhon,Lim, Yoong-Ho,Shim, Yhong-Hee,Ahn, Ji-yun,Kim, Hyoung-Choon,Chitwood, Dav Pharmaceutical Society of Japan 2007 BIOLOGICAL & PHARMACEUTICAL BULLETIN Vol.30 No.11
<P>The backbone structure of ginsenosides, active ingredients of <I>Panax ginseng</I>, is similar with that of sterol, especially cholesterol. <I>Caenorhabditis elegans</I> (<I>C. elegans</I>) is one of free living nematodes and is well-established animal model for biochemical and genetic studies. <I>C. elegans</I> cannot synthesize <I>de novo</I> cholesterol, although cholesterol is essential requirement for its growth and development. In the present study, we investigated the effects of ginseng total saponins (GTS) on the average brood size, growth, development, worm size, and life span of <I>C. elegans</I> in cholesterol-deprived and -fed medium. Cholesterol deprivation caused damages on normal growth, reproduction, and life span of worms throughout F1 to F3 generations. GTS supplement to cholesterol-deprived medium restored the growth, reproduction, and life span of worms as much as cholesterol alone-fed medium. GTS co-supplement to cholesterol-fed medium not only promoted worm reproduction but also induced bigger worms and faster growth than cholesterol-fed medium. In study to identify which ginsenosides are responsible for life span restoring effects of GTS, we found that ginsenoside Rc supplement not only restored life span of worms grown in cholesterol-deprived medium but also prolonged life span of worms grown in cholesterol-fed medium. Worms grown in medium supplemented with ginsenoside Rb<SUB>1</SUB> or Rc to cholesterol-deprived medium exhibited strong filipin staining, in which filipin forms tight and specific complexes with 3β-hydroxy sterols. These results show a possibility that ginsenosides could be utilized by <I>C. elegans</I> as a sterol substitute and further indicate that ginsenoside Rc is the component of <I>Panax ginseng</I> that prolongs the life span of <I>C. elegans</I>.</P>