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S450 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 [P-M.64] Influence of Endosulfan on Allergic Response in Mouse Sogo Nishimoto 1,2,, Satoko Atobe 2 , Masaaki Okabe 1,2 , Koichi Akiyama 3 , Yoshimi Kakinuma 2,3 , Takuya Sugahara 2,4 1 Center for Marine Environmental Studies, Ehime University, Japan 2 Faculty of Agriculture, Ehime University, Japan 3 Integrated Center of Science, Ehime University, Japan 4 Southern Ehime Fisheries Research Center, Ehime University, Japan Keywords: Endosulfan; Allergy; Immune response; Cytokine Many scientists have strong interest in studying the effects of agricultural chemicals on our health. We focused on endosul- fan, one of the agricultural chemicals, because the chemical was listed as new POPs candidates in 2005 (Stockholm Convention in 2005 “New POPs”). There are many reports concerning the poi- sonous effects of endosulfan on reproductive organs (Naqvi and Vaishnavi, 1993). However, there are only few reports on evaluation of immune toxicity of endosulfan. Hence, we investigated the effect of -endosulfan (one of the isomers) on immune system, especially allergy response. -endosulfan was orally administrated to ovalbumin (OVA)- challenged allergy mice for 27 days. Blood and spleen were collected on day 28, and spleen lymphocytes were isolated and cul- tured for 48 h. OVA-specific Ig levels and cytokine levels in serum and culture supernatant of spleen lymphocytes were measured by enzyme-linked immunosorbent assay (ELISA). The effect of - endosulfan on allergen-induced degranulation by rat basophilic leukemia cell line RBL-2H3 cells was examined. Oral administration of -endosulfan increased OVA-specific IgE and IgG1 levels in serum. -endosulfan also enhanced interleukin (IL)-4 level in serum, and IL-4 production by spleen lymphocytes. IL- 4 is a cytokine inducing IgE production by B cells. On the other hand, interferon (IFN)- level in serum was not affected by intake of - endosulfan. Allergy response is potentiated by imbalance between helper T sub-populations, Th1 and Th2 (Abbas et al., 1996). - endosulfan activated IL-4-producing Th2 cells. This suggests that -endosulfan inclines Th balance toward Th2 dominance, and allergy response is facilitated by imbalance of Th1/Th2. In addition, -endosulfan activated degranulation of RBL-2H3 cells. This result means that -endosulfan exaggerates allergy symptoms mediated by histamine and leukotriene in granules. Our findings suggest that intake of -endosulfan is a serious risk for our health. Acknowledgement This work was supported by Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Science, Sports, and Culture in Japan. References Naqvi, S.M., Vaishnavi, C., 1993. Bioaccumulative potential and toxicity of endosulfan insecticide to non-target animals. Comp. Biochem. Physiol. C. 105, 347–361. Abbas, A.K., et al., 1996. Functional diversity of helper T lymphocytes. Nature 383, 787–793. doi:10.1016/j.jbiotec.2010.09.652 [P-M.65] PCR clone of novel Staphylokinase gene from Staphylococcus aureus Abolfath Ebrahimi , Abdollah Ghasemian, Younes Ghasemi Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Islamic Republic of Iran Keywords: Gene cloning; Staphylokinase; Staphylococcus aureus; E.coli BL21(DE3) Introduction: Staphylokinase (SAK) is an extracellular protein. It is a potential therapeutic thrombolytic protein which converts the plasminogen, the inactive proenzyme of the fibrinolytic sys- tem, into plasmin. It is supposed to be effective in acute myocardial infarctions and it has a lower antigenicity, higher fibrin-specifity, and greater efficacy in comparison with streptokinase Methods: In the present work, the Staphylokinase from Staphy- lococcus aureus PTCC 1112 was cloned in pET15b and expressed in E.coli. Total genomic DNA were isolated and used for PCR amplifi- cation of the SAK gene. Desired sequence was amplified using the specific primers, which amplify a 500-bp of the structural SAK gene. The resulting PCR amplicon was ligated in the E.coli expres- sion vector pET15b. E.coli BL21(DE3) cells were transformed with the recombinant plasmid to get the expression strain Results: The amplified sequence was verified by DNA sequenc- ing and compared with other SAK genes on GenBank using BLAST tools. The staphylokinase activity was measured by using human plasminogen coupled chromogenic substrate assay by measuring the absorbance of released para-nitroaniline from S-2251 (D-Val- Leu-Lys-4-nitroanilide) at 405 nm. The recombinant enzyme has the same activity as wild-type staphylokinase. Molecular weight of recombinant protein estimated to be 15 kDa by SDS-PAGE Discussion: The characterization of new recombinant staphy- lokinase and the development of rapid, simple and effective production methods are not only of academic interest but also of clinical importance doi:10.1016/j.jbiotec.2010.09.653 [P-M.66] Preparation of chitosan/carboxymethyl dextran nanoparticles by polyelectrolyte complexation E.J. Lee 1 , S.A. Khan 2 , Y.-B. Kim 2 , K.-H. Lim 2,1 Kyungpook National University, Republic of Korea 2 Daegu University, Republic of Korea Keywords: Chitosan; Carboxymethyl dextran; Nanoparticle; Poly- electrolyte complexation The optimum condition was pursued for the preparation of the chitosan (polycation) /carboxymethyl dextran (CMD) (polyanion) polyelectrolyte-complex. Both the various mass-ratios of chitosan and CMD and the various pH of used chitosan solution were investi- gated to evaluate their effect on the turbidity of dispersion, the yield of dried mass, and the particle-size and the chemical structure of their polyelectrolyte-complex. The investigation was performed by the measurement of absorbance, the observation by SEM and the analysis of FT-IR spectrum. It was found that no turbidity appeared at all the mass-ratios studied with the chitosan solution of pH 2. With the chitosan solution of pH 5, a marked turbidity with no vis- ible aggregates was obtained at the chitosan-CMD mass-ratio of 1:4. However, at the same pH, the mass-ratio of 1:5 led to the for-

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S450 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-M.64]

Influence of Endosulfan on Allergic Response in Mouse

Sogo Nishimoto 1,2,∗, Satoko Atobe 2, Masaaki Okabe 1,2, KoichiAkiyama 3, Yoshimi Kakinuma 2,3, Takuya Sugahara 2,4

1 Center for Marine Environmental Studies, Ehime University, Japan2 Faculty of Agriculture, Ehime University, Japan3 Integrated Center of Science, Ehime University, Japan4 Southern Ehime Fisheries Research Center, Ehime University, JapanKeywords: Endosulfan; Allergy; Immune response; Cytokine

Many scientists have strong interest in studying the effectsof agricultural chemicals on our health. We focused on endosul-fan, one of the agricultural chemicals, because the chemical waslisted as new POPs candidates in 2005 (Stockholm Convention in2005 “New POPs”). There are many reports concerning the poi-sonous effects of endosulfan on reproductive organs (Naqvi andVaishnavi, 1993). However, there are only few reports on evaluationof immune toxicity of endosulfan. Hence, we investigated the effectof �-endosulfan (one of the isomers) on immune system, especiallyallergy response.

�-endosulfan was orally administrated to ovalbumin (OVA)-challenged allergy mice for 27 days. Blood and spleen werecollected on day 28, and spleen lymphocytes were isolated and cul-tured for 48 h. OVA-specific Ig levels and cytokine levels in serumand culture supernatant of spleen lymphocytes were measuredby enzyme-linked immunosorbent assay (ELISA). The effect of �-endosulfan on allergen-induced degranulation by rat basophilicleukemia cell line RBL-2H3 cells was examined.

Oral administration of �-endosulfan increased OVA-specific IgEand IgG1 levels in serum. �-endosulfan also enhanced interleukin(IL)-4 level in serum, and IL-4 production by spleen lymphocytes. IL-4 is a cytokine inducing IgE production by B cells. On the other hand,interferon (IFN)-� level in serum was not affected by intake of �-endosulfan. Allergy response is potentiated by imbalance betweenhelper T sub-populations, Th1 and Th2 (Abbas et al., 1996). �-endosulfan activated IL-4-producing Th2 cells. This suggests that�-endosulfan inclines Th balance toward Th2 dominance, andallergy response is facilitated by imbalance of Th1/Th2. In addition,�-endosulfan activated degranulation of RBL-2H3 cells. This resultmeans that �-endosulfan exaggerates allergy symptoms mediatedby histamine and leukotriene in granules. Our findings suggest thatintake of �-endosulfan is a serious risk for our health.

Acknowledgement

This work was supported by Grant-in-Aid for Young Scientists(B) from the Ministry of Education, Science, Sports, and Culture inJapan.

References

Naqvi, S.M., Vaishnavi, C., 1993. Bioaccumulative potential and toxicity of endosulfaninsecticide to non-target animals. Comp. Biochem. Physiol. C. 105, 347–361.

Abbas, A.K., et al., 1996. Functional diversity of helper T lymphocytes. Nature 383,787–793.

doi:10.1016/j.jbiotec.2010.09.652

[P-M.65]

PCR clone of novel Staphylokinase gene from Staphylococcusaureus

Abolfath Ebrahimi ∗, Abdollah Ghasemian, Younes Ghasemi

Department of Pharmaceutical Biotechnology, Faculty of Pharmacyand Pharmaceutical Sciences Research Center, Shiraz University ofMedical Sciences, Shiraz, Islamic Republic of IranKeywords: Gene cloning; Staphylokinase; Staphylococcus aureus;E.coli BL21(DE3)

Introduction: Staphylokinase (SAK) is an extracellular protein.It is a potential therapeutic thrombolytic protein which convertsthe plasminogen, the inactive proenzyme of the fibrinolytic sys-tem, into plasmin. It is supposed to be effective in acute myocardialinfarctions and it has a lower antigenicity, higher fibrin-specifity,and greater efficacy in comparison with streptokinase

Methods: In the present work, the Staphylokinase from Staphy-lococcus aureus PTCC 1112 was cloned in pET15b and expressed inE.coli. Total genomic DNA were isolated and used for PCR amplifi-cation of the SAK gene. Desired sequence was amplified using thespecific primers, which amplify a ∼500-bp of the structural SAKgene. The resulting PCR amplicon was ligated in the E.coli expres-sion vector pET15b. E.coli BL21(DE3) cells were transformed withthe recombinant plasmid to get the expression strain

Results: The amplified sequence was verified by DNA sequenc-ing and compared with other SAK genes on GenBank using BLASTtools. The staphylokinase activity was measured by using humanplasminogen coupled chromogenic substrate assay by measuringthe absorbance of released para-nitroaniline from S-2251 (D-Val-Leu-Lys-4-nitroanilide) at 405 nm. The recombinant enzyme hasthe same activity as wild-type staphylokinase. Molecular weight ofrecombinant protein estimated to be ∼ 15 kDa by SDS-PAGE

Discussion: The characterization of new recombinant staphy-lokinase and the development of rapid, simple and effectiveproduction methods are not only of academic interest but also ofclinical importance

doi:10.1016/j.jbiotec.2010.09.653

[P-M.66]

Preparation of chitosan/carboxymethyl dextran nanoparticlesby polyelectrolyte complexation

E.J. Lee 1, S.A. Khan 2, Y.-B. Kim 2, K.-H. Lim 2,∗

1 Kyungpook National University, Republic of Korea2 Daegu University, Republic of KoreaKeywords: Chitosan; Carboxymethyl dextran; Nanoparticle; Poly-electrolyte complexation

The optimum condition was pursued for the preparation of thechitosan (polycation) /carboxymethyl dextran (CMD) (polyanion)polyelectrolyte-complex. Both the various mass-ratios of chitosanand CMD and the various pH of used chitosan solution were investi-gated to evaluate their effect on the turbidity of dispersion, the yieldof dried mass, and the particle-size and the chemical structure oftheir polyelectrolyte-complex. The investigation was performed bythe measurement of absorbance, the observation by SEM and theanalysis of FT-IR spectrum. It was found that no turbidity appearedat all the mass-ratios studied with the chitosan solution of pH 2.With the chitosan solution of pH 5, a marked turbidity with no vis-ible aggregates was obtained at the chitosan-CMD mass-ratio of1:4. However, at the same pH, the mass-ratio of 1:5 led to the for-