&lt;b&gt;Immunocytochemical localization of lysenin, a novel protein isolated from the coelomic fluid of the earthworm &lt;i&gt;Eisenia &lt;/i&gt;&lt;/b&gt;&lt;b&gt;&lt;i&gt;foetida &lt;/i&gt;&lt;/b&gt;

Sekizawa, Yoshiyuki; Ohta, Naoshi; Natori, Shunji; Kobayashi, Hideshi

doi:10.2220/biomedres.17.327

Cited by 5 publications

(7 citation statements)

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“…We first examined the levels of sphingomyelin on the surface of proliferating C2C12 cells maintained in high-serum medium by probing fixed cells with 0.5 mg/ml lysenin. Subsequent immunostaining using a specific anti-lysenin antibody (Sekizawa et al 1996) showed that sphingomyelin on the surface of proliferating C2C12 cells was barely detectable (Figures 1A and 1B and quantified in Figure 2A). …”

Section: Resultsmentioning

confidence: 99%

“…For BrdU detection, cells were then treated with 3 N hydrochloric acid for 10 min at room temperature. Cells were then incubated with primary antibodies (mouse monoclonal anti-MyoD clone 5.8A [Dakocytomation; Carpinteria, CA]), anti-myogenin clone F5D (a gift from Dr. W. Wright at University of Texas), anti-sarcomeric myosin heavy chain (sMyHC) clone MF20, anti-BrdU clone G3G4 and anti-PAX7 (Developmental Studies Hybridoma Bank; Iowa City, IA), hamster monoclonal anti-Bcl-2 clone 3F11 (BD Pharmingen; San Diego, CA), and rabbit polyclonal antilysenin (Sekizawa et al 1996) (Peptide Institute Inc; Osaka, Japan). After washes in PBS, primary antibody binding was visualized with Alexa Fluor dye-conjugated secondary antibodies (Molecular Probes; Eugene, OR) for 30 min before washing and mounting in Fluoromount fluorescent mounting medium (DakoCytomation) containing 100 ng/ml 49,6-diamidino-2-phenylindole (DAPI) or Hoechst 33258.…”

Section: Lysenin Probing and Immunostainingmentioning

confidence: 99%

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Sphingomyelin Levels in the Plasma Membrane Correlate with the Activation State of Muscle Satellite Cells

Nagata

Kobayashi

Umeda

et al. 2006

J Histochem Cytochem.

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S U M M A R Y Satellite cells are responsible for postnatal growth, hypertrophy, and regeneration of skeletal muscle. They are normally quiescent, and must be activated to fulfill these functions, yet little is known of how this is regulated. As a first step in determining the role of lipids in this process, we examined the dynamics of sphingomyelin in the plasma membrane. Sphingomyelin contributes to caveolae/lipid rafts, which act to concentrate signaling molecules, and is also a precursor of several bioactive lipids. Proliferating or differentiated C2C12 muscle cells did not bind lysenin, a sphingomyelin-specific binding protein, but noncycling reserve cells did. Quiescent satellite cells also bound lysenin, revealing high levels of sphingomyelin in their plasma membranes. On activation, however, the levels of sphingomyelin drop, so that lysenin did not label proliferating satellite cells. Although most satellite cell progeny differentiate, others stop cycling, maintain Pax7, downregulate MyoD, and escape immediate differentiation. Importantly, many of these Pax7-positive/ MyoD-negative cells also regained lysenin binding on their surface, showing that the levels of sphingomyelin had again increased. Our observations show that quiescent satellite cells are characterized by high levels of sphingomyelin in their plasma membranes and that lysenin provides a novel marker of myogenic quiescence. (J Histochem Cytochem 54:375-384, 2006)

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Section: Resultsmentioning

confidence: 99%

Section: Lysenin Probing and Immunostainingmentioning

confidence: 99%

Sphingomyelin Levels in the Plasma Membrane Correlate with the Activation State of Muscle Satellite Cells

Nagata

Kobayashi

Umeda

et al. 2006

J Histochem Cytochem.

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“…The protein yielded a single band after SDS-polyacrylamide gel electrophoresis, and no contaminating protein bands were detected after staining the gel with Coomassie Brilliant Blue. An antiserum against lysenin was raised in male rabbits, as described previously (31), and its specificity was confirmed by immunoblotting analysis, which showed the antiserum recognized only one band of lysenin among all the proteins in the body fluid of E. foetida.…”

Section: Methodsmentioning

confidence: 98%

Lysenin, a Novel Sphingomyelin-specific Binding Protein

Yamaji

Sekizawa

Emoto

et al. 1998

Journal of Biological Chemistry

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319

332

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Lysenin, a novel 41-kDa protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of lysenin with vesicles containing sphingomyelin inhibited lysenin-induced hemolysis completely, whereas vesicles containing phospholipids other than sphingomyelin showed no inhibitory activity, suggesting that lysenin bound specifically to sphingomyelin on erythrocyte membranes. The specific binding of lysenin to sphingomyelin was confirmed by enzyme-linked immunosorbent assay, TLC immunostaining, and liposome lysis assay. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sphingomyelin analogs such as sphingosine, ceramide, and sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of sphingomyelin. Kinetic analysis of the lysenin-sphingomyelin interaction by surface plasmon resonance measurements using BIAcore TM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (k on ‫؍‬ 3.2 ؋ 10 4 M ؊1 s ؊1 ) and dissociated extremely slowly (k off ‫؍‬ 1.7 ؋ 10 ؊4 s ؊1 ), giving a low dissociation constant (K D ‫؍‬ 5.3 ؋ 10 ؊9 M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lysenin bound to the membrane, whereas it did not change the kinetic parameters of the lysenin-membrane interaction, suggesting that lysenin specifically recognized sphingomyelin and cholesterol incorporation changed the topological distribution of sphingomyelin in the membranes, thereby increasing the accessibility of sphingomyelin to lysenin. Immunofluorescence staining of fibroblasts derived from a patient with Niemann-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by digitonin treatment. Preincubation of lysenin with vesicles containing sphingomyelin abolished lysenin immunostaining. This study demonstrated that lysenin bound specifically to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biological membranes.Sphingomyelin, N-acylsphingosine-1-phosphorylcholine, is a major lipid constituent of animal cell membranes, accounting for up to 60 mol % of the total phospholipid content of some cells, such as sheep erythrocytes (1). In addition to its role as a structural component of membranes, recent studies have suggested the roles of sphingomyelin in various intracellular signaling pathways (2-6). A sphingomyelin cycle has been proposed in which extracellular agents such as tumor necrosis factor ␣, ␥-interferon, and interleukin 1 activate sphingomyelinase, resulting in the production of ceramide which serves as a second messenger mediating the action of these extracellular ligands (4 -6). Sphingomyelin was also proposed to be enriched in an organized membrane domain, caveolae, which contains cholesterol, glyco...

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“…Sections treated with the lysenin antiserum that had been incubated with lysenin showed no evidence of immunoreactivity in all tissues examined. We reported previously that the large coelomocytes were immunoreactive to the lysenin antiserum (Sekizawa et al 1996b).…”

Section: Immunohistochemical Stainingmentioning

confidence: 88%

“…Immunohistochemical staining for light microscopy was performed as described by Sekizawa et al (1996b), with some modifications, using the same lysenin antiserum as used in the cited study. In brief, earthworms were cut into four pieces and fixed in 4% paraformaldehyde for 18 h. After fixation, the tissues were dehydrated through an ethanol series, cleared in xylene, and embedded in paraffin.…”

Section: Immunohistochemical Stainingmentioning

confidence: 99%

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida

Ohta

Shioda

Sekizawa

et al. 2000

Cell and Tissue Research

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Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.

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<b>Immunocytochemical localization of lysenin, a novel protein isolated from the coelomic fluid of the earthworm <i>Eisenia </i></b><b><i>foetida </i></b>

Cited by 5 publications

References 0 publications

Sphingomyelin Levels in the Plasma Membrane Correlate with the Activation State of Muscle Satellite Cells

Sphingomyelin Levels in the Plasma Membrane Correlate with the Activation State of Muscle Satellite Cells

Lysenin, a Novel Sphingomyelin-specific Binding Protein

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida

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