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-

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S451

mation of big visible aggregates with complete phase-separation.With the chitosan solution of pH 6, the chitosan-CMD mass-ratioof 1:3, 1:4 and 1:5, produced large visible aggregates. Neverthelessthe lower mass-ratio of 1:2 at this pH yielded a turbid suspensionwith no visible aggregates. These results suggest that at higher pHof chitosan solution, the relatively lower quantity of CMD yieldsa turbid suspension and the higher quantity of CMD consequentlyleads to big visible aggregates. The FT-IR spectrum of chitosan-CMDpolyelectrolyte-complex explains that an electrostatic interactionhas occurred between the carboxylic groups of CMD and the aminogroups of chitosan, and the extent of their interaction increaseswith increasing the pH of chitosan solution.

doi:10.1016/j.jbiotec.2010.09.654

[P-M.67]

Copper affects spermatozoa motility, morphology and cellmembrane integrity in rabbits in vitro

Shubhadeep Roychoudhury 1,∗, Jozef Bulla 1, Peter Massanyi 1,Manabendra Dutta Choudhury 2

1 Slovak University of Agriculture, Slovakia2 Assam University, IndiaKeywords: Copper toxicity; Spermatozoa motility; Morphology;Membrane integrity

This study focused on effects of copper on spermatozoa motility,morphology and cell membrane integrity in rabbits in vitro, using anarrow spectrum of concentrations (3.57 till 4.85 �g CuSO4/mL; E<D < C < B < A < Max) based on already reported wider range. Pooledrabbit semen was cultured in vitro with copper (5% CuSO4.5H2O)and diluted to experimental concentrations. Computer assistedsemen analyzer (CASA) detected decrease of total motility with inconcentration range of 3.70 to 4.85 �g CuSO4/mL. Similar trend wasexhibited for progressive motility. Distance parameters showedno change at Time 0; parameters declined in all concentrationswith increasing time up to 1 h, but exhibited a somewhat recov-ering trend after 2 h of culture in lower concentrations (3.77 �gCuSO4/mL in group B to 3.57 �g CuSO4/mL in group E). Similar trendwas noted for velocity parameters. This indicates that a sort of stresstolerance developed at lower concentrations. Other parameters(straightness, linearity, wobble, amplitude of lateral head displace-ment, beat cross frequency) showed a general trend of decrease athigh concentrations. Spermatozoa morphology analysis after 2 h ofcopper culture revealed significantly higher occurrence of morpho-logical abnormality at highest concentration. Annexin V analysisfor detection of spermatozoa with disordered membranes showedhigher occurrence of positive spermatozoa in copper exposedgroups, 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, toxiceffect confirmed by analyses of spermatozoa morphology andmembrane integrity. Data indicates adverse effects of copper as arisk factor of environment on spermatozoa movement, morphologyand membrane integrity, and its potential to cause male infertilityin relatively low doses.

doi:10.1016/j.jbiotec.2010.09.655

[P-M.68]

A Rapid Antimicrobial testing by using the Cr-Cu-N nanocom-posite 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, Taiwan2 Tungnan University, Taiwan3 Graduate School of Environmental Education & Resources, TaipeiMunicipal University of Education, TaiwanKeywords: Antibacterial tests; Minimum inhibitory concentrations(MICs); Copper; NANOCOMPOSITE thin film

Nosocomial infection by bacteria is a serious problem in theimplanted medical devices. It is reported that the adhesion ofbacteria to a biomaterial surface is the main reason for theimplant-associated infections. The antibacterial effects of copperare well documented in literatures, also effective on the epidemicmeticillin-resistant S. aureus. Addition of Cu into the transitionmetal nitride films (CrN) to form non-toxic nanocomposite thinfilms providing adequate bactericidal effects and long-term releas-ing properties have been done in previous work. The antimicrobialeffects of the CrCuN materials were determined not only by theCu contents, also by which pathogens were selected. In this study,the CrCuN nanocomposite thin films were deposited on metal sub-strates with different Cu contents using bipolar asymmetry reactivepulsed DC sputtering system. The thin films with gradient coppercontent ranging from 4 to 20 at.% were achieved. These fabricatedmaterials were expected to determine their minimum inhibitoryconcentrations (MICs) on different species of pathogens by a rapidmethod, since the E-test is an antimicrobial agent gradient-coatedplastic test strip which allows for quantitative antimicrobial sus-ceptibility testing on agar media. The MICs were estimated after24 hr incubation on the measurement of the elliptical zone ofgrowth inhibition due to the copper ion which diffused from thespecimens with gradient copper content. It was observed that theinterspaces 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. Theinterspaces of the antibacterial zones on various species were pro-portional to the antibacterial rates under different copper contentranges of thin films. The nanocomposite thin films with gradientcopper content are useful for rapid screening antimicrobial sus-ceptibility testing on various kinds of bacteria.

doi:10.1016/j.jbiotec.2010.09.656

[P-M.69]

Purification and characterization of antimicrobial peptidesfrom fleshfly Neobellieria bullata

Martina Mackova 1,2,∗, Tomas Macek 1,2, Miloslav Sanda 1,3,Zdenek Voburka Voburka 3

1 ICT Prague, Czech Republic2 Joint Laboratory of ICT and IOCB, Czech Republic3 Institute of Organic Chemistry and Biochemistry, Czech Academy ofSciences, Czech RepublicKeywords: Antimicrobial peptide; Fleshfly

Microbiological research is focused on solution of pathogenresistance problem. The number of resistant pathogenic microor-ganisms has increased in last years. In the future there will be fewerpossibilities to cure multiresistance infections by existing antibi-otics. One of the potential solutions seems to be short cationicpeptides synthesised as a part of innate immunity. In our project