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Expression and characterization of α-methylacyl CoA racemase from Anisakis simplex larvae.

Kim BJ, Kim SM, Cho MK, Yu HS, Lee YS, Cha HJ, Ock M - Korean J. Parasitol. (2012)

Bottom Line: A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species.Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr).The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Kosin University College of Medicine, Busan 602-703, Korea.

ABSTRACT
Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as α-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

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Expression of the Amacr in Anisakis simplex larvae. The Amacr product was observed primarily in the ventriculus. (A) Control staining of larval ventriculus (arrows) with normal mouse serum (×40). (B) Specifically stained ventriculus (arrows). The L3 were incubated at 24℃ for 24 hr (×40). (C) Larval Amacr immunostaining incubated for 48 hr also represents the same localization as 24 hr larvae. Ventriculus (arrows) (×100). Immunohistochemical study showed that the Amacr product was found specifically in the ventriculus of the larvae.
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Figure 6: Expression of the Amacr in Anisakis simplex larvae. The Amacr product was observed primarily in the ventriculus. (A) Control staining of larval ventriculus (arrows) with normal mouse serum (×40). (B) Specifically stained ventriculus (arrows). The L3 were incubated at 24℃ for 24 hr (×40). (C) Larval Amacr immunostaining incubated for 48 hr also represents the same localization as 24 hr larvae. Ventriculus (arrows) (×100). Immunohistochemical study showed that the Amacr product was found specifically in the ventriculus of the larvae.

Mentions: The expression of the Anisakis Amacr was constitutively high under all conditions tested, suggesting that a large amount of Amacr gene expression in A. simplex L3 is required over a long period for the larval survival (Fig. 3). The expression of recombinant A. simplex Amacr protein was confirmed with SDS-PAGE. The GST-fusion protein revealed a 73 kDa band on the gel (Fig. 4). The recombinant Amacr protein showed a reactive band against Amacr antibody, a rabbit polyclonal to AMCR (Abcam) (Fig. 5). Immunohistochemical analysis showed that the Amacr protein was mainly localized at the ventriculus of A. simplex larvae, regardless of different time points (24 and 48 hr) (Fig. 6).


Expression and characterization of α-methylacyl CoA racemase from Anisakis simplex larvae.

Kim BJ, Kim SM, Cho MK, Yu HS, Lee YS, Cha HJ, Ock M - Korean J. Parasitol. (2012)

Expression of the Amacr in Anisakis simplex larvae. The Amacr product was observed primarily in the ventriculus. (A) Control staining of larval ventriculus (arrows) with normal mouse serum (×40). (B) Specifically stained ventriculus (arrows). The L3 were incubated at 24℃ for 24 hr (×40). (C) Larval Amacr immunostaining incubated for 48 hr also represents the same localization as 24 hr larvae. Ventriculus (arrows) (×100). Immunohistochemical study showed that the Amacr product was found specifically in the ventriculus of the larvae.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375458&req=5

Figure 6: Expression of the Amacr in Anisakis simplex larvae. The Amacr product was observed primarily in the ventriculus. (A) Control staining of larval ventriculus (arrows) with normal mouse serum (×40). (B) Specifically stained ventriculus (arrows). The L3 were incubated at 24℃ for 24 hr (×40). (C) Larval Amacr immunostaining incubated for 48 hr also represents the same localization as 24 hr larvae. Ventriculus (arrows) (×100). Immunohistochemical study showed that the Amacr product was found specifically in the ventriculus of the larvae.
Mentions: The expression of the Anisakis Amacr was constitutively high under all conditions tested, suggesting that a large amount of Amacr gene expression in A. simplex L3 is required over a long period for the larval survival (Fig. 3). The expression of recombinant A. simplex Amacr protein was confirmed with SDS-PAGE. The GST-fusion protein revealed a 73 kDa band on the gel (Fig. 4). The recombinant Amacr protein showed a reactive band against Amacr antibody, a rabbit polyclonal to AMCR (Abcam) (Fig. 5). Immunohistochemical analysis showed that the Amacr protein was mainly localized at the ventriculus of A. simplex larvae, regardless of different time points (24 and 48 hr) (Fig. 6).

Bottom Line: A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species.Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr).The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Kosin University College of Medicine, Busan 602-703, Korea.

ABSTRACT
Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as α-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

Show MeSH
Related in: MedlinePlus