Korean Journal of Veterinary Research (대한수의학회지)

The Korean Society of Veterinary Science (대한수의학회)
권호 표지

Comparison of DNase activities from excretory/secretory productsof Haemonchus contortus fenbendazole-resistantand -susceptible isolates

Fenbendazole에 저항성과 감수성을 지닌 염전위충의 분비배설물에서의 DNase 활성 비료

  • Kwak, Dongmi (Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University)
  • 곽동미 (워싱턴주립대학교 수의과대학)
  • Accepted : 2004.06.14
  • Published : 2004.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Change in ${\beta}$-tubulin nucleic acid and protein sequences was the only known difference between Haemonchus contortus fenbendazole (FBZ)-resistant and -susceptible isolates. This change was sufficient to determine the pathologic effect induced by FBZ treatment. This research was initiated to investigate further differences from these two isolates. Since ${\beta}$-tubulin is involved in formation of microtubule, which has functions in secretory vesicle transport, DNase activities from excretory/secretory products (ESP) of the two isolates were compared, based on pH, sensitivity to DNase inhibitors, molecular masses and production of 3'-OH. The most significant difference detected was that a 38.5 kDa DNase activity was identified from ESP of H. contortus FBZ-susceptible isolates but not from those of H. contortus FBZ-resistant isolates. However, it was shown that the 38.5 kDa DNase is expressed with similar level of activity in intestine and whole worm of H. contortus FBZ-resistant and -susceptible isolates. This result demonstrated that the secretory transport pathway of the 38.5 kDa DNase was inhibited by unknown mechanisms, which may be related with ${\beta}$-tubulin sequence change in FBZ-resistant isolates. Other DNases of 34, 36 and 37 kDa were detected from ESP of both H. contortus FBZ-resistant and -susceptible isolates. Overall DNase activities found from ESP of these two isolates were not inhibited by 10 mM EDTA at pH 5.0, but largely inhibited by pH 7.0. In addition, DNase activities in two isolates produced DNA fragments with mixtures of 3'- hydroxyls (OH) and 3'-phosphates (P) at each pH although the 3'-end labeling ratios at pH 5.0 and 7.0 were shown different. Identification of inhibition of the 38.5 kDa DNase secretion in FBZ-resistant isolates suggests existence of further differences, in addition to ${\beta}$-tubulin sequence change, in two isolates. This shows complex effect of FBZ on H. contortus biological mechanisms.

Keywords

References

  1. Borgers, M., De Nollin, S., De Brabander, M. and Thienpont, D. Influence of the anthelmintic mebendazole on microtubules and intracellular organelle movementin nematode intestinal cells. Am. J. Vet. Res. 1975, 36, 1153-1166
  2. Jasmer, D. P., Yao, C., Rehman, A. and Johnson, S. Multiple lethal effects induced by a benzimidazole anthelmintic in the anterior intestine of the nematodeHaemonchus contortus. Mol. Biochem. Parasitol. 2000, 105, 81-90
  3. Kwa, M. S., Veenstra, J. G. and Roos, M. H. Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200in beta-tubulin isotype 1. Mol. Biochem. Parasitol. 1994, 63, 299-303
  4. Kwa, M. S., Veenstra, J. G., Van Dijk, M. and Roos, M. H. Beta-tubulin genes from the parasitic nematode Haemonchus contortus modulate drug resistance inCaenorhabditis elegans. J. Mol. Biol. 1995, 246, 500-510
  5. Kwak, D. and Jasmer, D. P. Non-classic characteristics define prominent DNase activities from the intestine and other tissues of Haemonchus contortus. Exp. Parasitol. 2003, 104, 131-139
  6. Lubega, G. W. and Prichard, R. K. Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy. Exp. Parasitol. 1991, 73, 203-213
  7. Prats, E., Noel, M., Letourneau, J., Tiranti, V., Vaque, J., Debon, R., Zeviani, M., Cornudella, L. and Ruiz-Carrillo, A. Characterization and expressionof the mouse endonuclease G gene. DNA Cell Biol. 1997, 16, 1111-1122
  8. Shak, S., Capon, D. J., Hellmiss, R., Marsters, S. A. and Baker, C. L. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc. Natl. Acad. Sci. USA. 1990, 87, 9188-9192
  9. Shiokawa, D. and Tanuma, S. Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase gamma. Biochem. J. 1998, 332,713-720
  10. Shiokawa, D., Shika, Y. and Tanuma, S. Identification of two functional nuclear localization signals in DNase gamma and their roles in its apoptotic DNase activity. Biochem. J. 2003, 376, 377-381
  11. Shompole, S., Yao, C., Cheng, X., Knox, D., Johnson, S. and Jasmer, D. P. Distinct characteristics of two intestinal protein compartments discriminated by using fenbendazole and a benzimidazole resistant isolate of Haemonchus contortus. Exp. Parasitol. 2002, 101, 200-209
  12. Yasuda, T., Takeshita, H., Iida, R., Nakajima, T., Hosomi, O., Nakashima, Y. and Kishi, K. Molecular cloning of the cDNA encoding human deoxyribonucleaseII. J. Biol. Chem. 1998, 273, 2610-2616