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Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

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Chicken gizzard homogenates were separated by 10%  SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to either SMαA (lanes a and b) or the 1E12 antigen (lanes  c and d). Gizzard homogenates were prepared by extraction in  TBS alone (lanes a and c) or reextraction of the TBS-insoluble  pellet in 6 M urea/TBS (lanes b and d). Immunoreactivity was determined by enhanced chemiluminescence techniques. 1E12 ascites were incubated with 0.5% SDS before incubation with the  membrane and used at a final dilution of 1:1,000. 1E12 does not  immunoreact with actin, but is strongly immunoreactive with a  band migrating at Mr = 100,000 (arrow). In addition, a doublet  migrating ahead of actin on the gel is often observed to be immunoreactive (lane d).
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Figure 4: Chicken gizzard homogenates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to either SMαA (lanes a and b) or the 1E12 antigen (lanes c and d). Gizzard homogenates were prepared by extraction in TBS alone (lanes a and c) or reextraction of the TBS-insoluble pellet in 6 M urea/TBS (lanes b and d). Immunoreactivity was determined by enhanced chemiluminescence techniques. 1E12 ascites were incubated with 0.5% SDS before incubation with the membrane and used at a final dilution of 1:1,000. 1E12 does not immunoreact with actin, but is strongly immunoreactive with a band migrating at Mr = 100,000 (arrow). In addition, a doublet migrating ahead of actin on the gel is often observed to be immunoreactive (lane d).

Mentions: The 1E12 IgM mAb proved to be unsuitable for affinity chromatography and immunoprecipitation. However, 1E12 can be used for immunoblotting if, before incubation with electroblotted antigen, this IgM antibody is pretreated with a buffered solution containing dilute SDS (Hungerford et al., 1996). Using this immunoblotting method, we probed extracts of adult chicken gizzard (an abundant source of smooth muscle antigens) for the 1E12 antigen. When homogenized gizzard tissue was extracted with TBS, a 100-kD band was detected (Fig. 4, lane c). If the resulting pellet was reextracted with the same buffer containing 6 M urea, additional 1E12 antigen was detected (Fig. 4, lane d). This suggested that at least some of the 1E12 antigen remained insoluble at neutral pH and normal ionic strength. Lanes a and b show the results of probing two similar samples with antibodies to SMαA. In addition to the 100-kD polypeptide band recognized by 1E12, a doublet at ∼40 kD was detected in some preparations but not in others. This doublet migrates well ahead of SMαA (compare lanes a and b with c and d). Also, based on the urea extraction data, the 1E12 antigen appears to be less soluble than SMαA.


Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

Chicken gizzard homogenates were separated by 10%  SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to either SMαA (lanes a and b) or the 1E12 antigen (lanes  c and d). Gizzard homogenates were prepared by extraction in  TBS alone (lanes a and c) or reextraction of the TBS-insoluble  pellet in 6 M urea/TBS (lanes b and d). Immunoreactivity was determined by enhanced chemiluminescence techniques. 1E12 ascites were incubated with 0.5% SDS before incubation with the  membrane and used at a final dilution of 1:1,000. 1E12 does not  immunoreact with actin, but is strongly immunoreactive with a  band migrating at Mr = 100,000 (arrow). In addition, a doublet  migrating ahead of actin on the gel is often observed to be immunoreactive (lane d).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139835&req=5

Figure 4: Chicken gizzard homogenates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to either SMαA (lanes a and b) or the 1E12 antigen (lanes c and d). Gizzard homogenates were prepared by extraction in TBS alone (lanes a and c) or reextraction of the TBS-insoluble pellet in 6 M urea/TBS (lanes b and d). Immunoreactivity was determined by enhanced chemiluminescence techniques. 1E12 ascites were incubated with 0.5% SDS before incubation with the membrane and used at a final dilution of 1:1,000. 1E12 does not immunoreact with actin, but is strongly immunoreactive with a band migrating at Mr = 100,000 (arrow). In addition, a doublet migrating ahead of actin on the gel is often observed to be immunoreactive (lane d).
Mentions: The 1E12 IgM mAb proved to be unsuitable for affinity chromatography and immunoprecipitation. However, 1E12 can be used for immunoblotting if, before incubation with electroblotted antigen, this IgM antibody is pretreated with a buffered solution containing dilute SDS (Hungerford et al., 1996). Using this immunoblotting method, we probed extracts of adult chicken gizzard (an abundant source of smooth muscle antigens) for the 1E12 antigen. When homogenized gizzard tissue was extracted with TBS, a 100-kD band was detected (Fig. 4, lane c). If the resulting pellet was reextracted with the same buffer containing 6 M urea, additional 1E12 antigen was detected (Fig. 4, lane d). This suggested that at least some of the 1E12 antigen remained insoluble at neutral pH and normal ionic strength. Lanes a and b show the results of probing two similar samples with antibodies to SMαA. In addition to the 100-kD polypeptide band recognized by 1E12, a doublet at ∼40 kD was detected in some preparations but not in others. This doublet migrates well ahead of SMαA (compare lanes a and b with c and d). Also, based on the urea extraction data, the 1E12 antigen appears to be less soluble than SMαA.

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Show MeSH