carboxymethyl dextran nanoparticles by polyelectrolyte complexation

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

[P-M.64]

[P-M.65]

Influence of Endosulfan on Allergic Response in Mouse

PCR clone of novel Staphylokinase gene from Staphylococcus aureus

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

Abolfath Ebrahimi ∗ , Abdollah Ghasemian, Younes Ghasemi

Center for Marine Environmental Studies, Ehime University, Japan 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

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)

Many scientists have strong interest in studying the effects of agricultural chemicals on our health. We focused on endosulfan, 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 poisonous 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 cultured 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. IL4 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.

Introduction: Staphylokinase (SAK) is an extracellular protein. It is a potential therapeutic thrombolytic protein which converts the plasminogen, the inactive proenzyme of the fibrinolytic system, 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 Staphylococcus aureus PTCC 1112 was cloned in pET15b and expressed in E.coli. Total genomic DNA were isolated and used for PCR amplification 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 expression 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 sequencing 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-ValLeu-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 staphylokinase and the development of rapid, simple and effective production methods are not only of academic interest but also of clinical importance

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

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 Daegu University, Republic of Korea Keywords: Chitosan; Carboxymethyl dextran; Nanoparticle; Polyelectrolyte complexation 2

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

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 investigated 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 visible 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-

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

mation of big visible aggregates with complete phase-separation. With the chitosan solution of pH 6, the chitosan-CMD mass-ratio of 1:3, 1:4 and 1:5, produced large visible aggregates. Nevertheless the lower mass-ratio of 1:2 at this pH yielded a turbid suspension with no visible aggregates. These results suggest that at higher pH of chitosan solution, the relatively lower quantity of CMD yields a turbid suspension and the higher quantity of CMD consequently leads to big visible aggregates. The FT-IR spectrum of chitosan-CMD polyelectrolyte-complex explains that an electrostatic interaction has occurred between the carboxylic groups of CMD and the amino groups of chitosan, and the extent of their interaction increases with increasing the pH of chitosan solution. doi:10.1016/j.jbiotec.2010.09.654 [P-M.67] Copper affects spermatozoa motility, morphology and cell membrane integrity in rabbits in vitro Shubhadeep Roychoudhury 1,∗ , Jozef Bulla 1 , Peter Massanyi 1 , Manabendra Dutta Choudhury 2 1

Slovak University of Agriculture, Slovakia Assam University, India Keywords: Copper toxicity; Spermatozoa motility; Morphology; Membrane integrity 2

This study focused on effects of copper on spermatozoa motility, morphology and cell membrane integrity in rabbits in vitro, using a narrow spectrum of concentrations (3.57 till 4.85 ␮g CuSO4 /mL; E< D < C < B < A < Max) based on already reported wider range. Pooled rabbit semen was cultured in vitro with copper (5% CuSO4.5H2O) and diluted to experimental concentrations. Computer assisted semen analyzer (CASA) detected decrease of total motility with in concentration range of 3.70 to 4.85 ␮g CuSO4 /mL. Similar trend was exhibited for progressive motility. Distance parameters showed no change at Time 0; parameters declined in all concentrations with increasing time up to 1 h, but exhibited a somewhat recovering trend after 2 h of culture in lower concentrations (3.77 ␮g CuSO4 /mL in group B to 3.57 ␮g CuSO4 /mL in group E). Similar trend was noted for velocity parameters. This indicates that a sort of stress tolerance developed at lower concentrations. Other parameters (straightness, linearity, wobble, amplitude of lateral head displacement, beat cross frequency) showed a general trend of decrease at high concentrations. Spermatozoa morphology analysis after 2 h of copper culture revealed significantly higher occurrence of morphological abnormality at highest concentration. Annexin V analysis for detection of spermatozoa with disordered membranes showed higher occurrence of positive spermatozoa in copper exposed groups, suggesting alterations in anterior part of head (acrosome) and in connection segment (mid-piece). We report the lowest possible toxic concentration of copper (3.70 ␮g CuSO4 /mL) to spermatozoa in relation to motility, toxic effect confirmed by analyses of spermatozoa morphology and membrane integrity. Data indicates adverse effects of copper as a risk factor of environment on spermatozoa movement, morphology and membrane integrity, and its potential to cause male infertility in relatively low doses. doi:10.1016/j.jbiotec.2010.09.655

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[P-M.68] A Rapid Antimicrobial testing by using the Cr-Cu-N nanocomposite thin films with gradient copper content J.W. Lee 1 , Y.J. Chang 3,∗ , G.J. Weng 2 , J.T. Chen 2 , C.L. Li 2 1

Mingchi University of Technology, Taiwan Tungnan University, Taiwan 3 Graduate School of Environmental Education & Resources, Taipei Municipal University of Education, Taiwan Keywords: Antibacterial tests; Minimum inhibitory concentrations (MICs); Copper; NANOCOMPOSITE thin film 2

Nosocomial infection by bacteria is a serious problem in the implanted medical devices. It is reported that the adhesion of bacteria to a biomaterial surface is the main reason for the implant-associated infections. The antibacterial effects of copper are well documented in literatures, also effective on the epidemic meticillin-resistant S. aureus. Addition of Cu into the transition metal nitride films (CrN) to form non-toxic nanocomposite thin films providing adequate bactericidal effects and long-term releasing properties have been done in previous work. The antimicrobial effects of the CrCuN materials were determined not only by the Cu contents, also by which pathogens were selected. In this study, the CrCuN nanocomposite thin films were deposited on metal substrates with different Cu contents using bipolar asymmetry reactive pulsed DC sputtering system. The thin films with gradient copper content ranging from 4 to 20 at.% were achieved. These fabricated materials were expected to determine their minimum inhibitory concentrations (MICs) on different species of pathogens by a rapid method, since the E-test is an antimicrobial agent gradient-coated plastic test strip which allows for quantitative antimicrobial susceptibility testing on agar media. The MICs were estimated after 24 hr incubation on the measurement of the elliptical zone of growth inhibition due to the copper ion which diffused from the specimens with gradient copper content. It was observed that the interspaces of the antibacterial zones are around 1-4 mm, 1-2 mm, and 1-10 mm for E. coli, P. aeruginosa and S. aureus, respectively. The interspaces of the antibacterial zones on various species were proportional to the antibacterial rates under different copper content ranges of thin films. The nanocomposite thin films with gradient copper content are useful for rapid screening antimicrobial susceptibility testing on various kinds of bacteria. doi:10.1016/j.jbiotec.2010.09.656 [P-M.69] Purification and characterization of antimicrobial peptides from fleshfly Neobellieria bullata Martina Mackova 1,2,∗ , Tomas Zdenek Voburka Voburka 3

Macek 1,2 , Miloslav

Sanda 1,3 ,

1

ICT Prague, Czech Republic Joint Laboratory of ICT and IOCB, Czech Republic 3 Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Czech Republic Keywords: Antimicrobial peptide; Fleshfly 2

Microbiological research is focused on solution of pathogen resistance problem. The number of resistant pathogenic microorganisms has increased in last years. In the future there will be fewer possibilities to cure multiresistance infections by existing antibiotics. One of the potential solutions seems to be short cationic peptides synthesised as a part of innate immunity. In our project