ALBERT

Description:    Antitumor agents are used in therapy against many forms of human cancer. One of these is mitomycin-C (MMC). As with many agents, it can interact with biological molecules and can induce genetic hazards in non-tumor cells. One of the possible approaches to protect DNA from this damage is to supply antioxidants that can remove free radicals produced by antitumor agents. Lipoic acid (LA) is known as one of the most powerful antioxidants. The aim of this study was to investigate antigenotoxic effects of LA against MMC induced chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronucleus (MN) formation in human lymphocytes. Lymphocytes were treated with 0.2 μg MMC/heparinized mL for 48 h. Three different concentrations (0.5, 1, 2 μg/mL) of LA were used together with MMC in three different applications; 1 h pre-treatment, simultaneous treatment and 1 h post-treatment. A negative, a positive and a solvent control were also included. In all the cultures treated with MMC + LA, the frequency of abnormal cells and CA/cell significantly decreased compared to MMC. Statistically significant reduction was also observed in SCE/cell and MN frequencies in all treatments. These results demonstrated anticlastogenic and antimutagenic effects of LA against MMC induced genotoxicity. LA showed the most efficient effect during 1 h pretreatment. On the other hand, MMC + LA treatments induced significant reduction in mitotic index than that of MMC treatment alone. These results are encouraging that LA can be a possible chemopreventive agent in tumorigenesis in both cancer patients and in health care persons handling anti-cancer drugs. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9504-8 Authors Fatma Unal, Department of Biology, Science Faculty, Gazi University, Ankara, Turkey Gokce Taner, Department of Biology, Science Faculty, Gazi University, Ankara, Turkey Deniz Yuzbasioglu, Department of Biology, Science Faculty, Gazi University, Ankara, Turkey Serkan Yilmaz, Department of Midwifery, Faculty of Health Sciences, Ankara University, Ankara, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps. Content Type Journal Article Category Brief Report Pages 1-10 DOI 10.1007/s10616-012-9509-3 Authors Iria Maria Gomez-Touriño, CIMUS (Department of Physiology), School of Medicine, University of Santiago de Compostela, Avd. Barcelona, 15782 Santiago de Compostela, Spain Ana Senra, CIMUS (Department of Physiology), School of Medicine, University of Santiago de Compostela, Avd. Barcelona, 15782 Santiago de Compostela, Spain Francisco Garcia, CIMUS (Department of Physiology), School of Medicine, University of Santiago de Compostela, Avd. Barcelona, 15782 Santiago de Compostela, Spain Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The present study aims to investigate the heptonephro-protective effect of grape seeds proanthocyanidin extract (GSPE) against the risks induced by gibberellic acid (GA3) in male rats. The results recorded that GA3 caused a significant increase in total lipids, total cholesterol, triglycerides and LDL-C levels in serum, concomitant with a significant decrease in serum HDL-C. A significant increase in serum AST, ALT, urea and creatinine, while, a significant decrease in total protein content in serum was observed in rats given GA3. Hepatic and renal lipid peroxidation product (MDA) was significantly increased, meanwhile, total antioxidant capacity (TAC), glutathione, and catalase levels were significantly decreased. In addition, there was a negative change in liver structure including dilatation in the central veins with degeneration of endothelium cells and cellular injury around the veins as well as in the kidney structure such as lesion in both glomeruli and tubules, detachment of the Malpighian corpuscles from the Bowman’s capsule’s epithelium, shrinkage in the glomerular capillary network. However, almost all of these adverse effects seemed to be ameliorated by oral administration of GSPE with GA3 to rats for 2 month indicating the protective effect of grape seeds GSPE on GA3 induced oxidative stress in rats. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9506-6 Authors Hanaa A. Hassan, Faculty of Science, Zoology Department, Mansoura University, Mansoura, Egypt Maisaa M. Al-Rawi, Biology Department, Practical Science College, Umm Al-Qura University, Mecca, Saudi Arabia Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9499-1 Authors Wenjing Zhang, The State Key Laboratory of Quality Research in Chinese Medicine, Faculty of Chinese Medicine, Macau University of Science and Technology, Taipa Macau, China Yan Lan, The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, China Qilai Huang, The State Key Laboratory of Quality Research in Chinese Medicine, Faculty of Chinese Medicine, Macau University of Science and Technology, Taipa Macau, China Zichun Hua, The State Key Laboratory of Quality Research in Chinese Medicine, Faculty of Chinese Medicine, Macau University of Science and Technology, Taipa Macau, China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phase and M4: G2/M phase of cell cycle in brain tissue ( p  〈 0.05). Significant increase in % of apoptosis and P53 protein expression as apoptotic biomarkers were coincided with significant decrease in Bcl 2 as an anti-apoptotic marker. The biochemical analysis recorded a significant decrease in the levels of reduced glutathione, superoxide dismutase, deoxyribonucleic acid (DNA) and ribonucleic acid contents. Moreover, numerous histopathological alterations were detected in brain tissues of gamma irradiated mice such as signs of chromatolysis in pyramidal cells of cortex, nuclear vacuolation, numerous apoptotic cell, and neural degeneration. On the other hand, gamma irradiated mice pretreated with Mentha extract showed largely an improvement in all the above tested parameters through a homeostatic state for the content of brain apoptosis and stabilization of DNA cycle with a distinct improvement in cell cycle analysis and antioxidant defense system. Furthermore, the aforementioned effects of Mentha extracts through down-regulation of P53 expression and up-regulation of Bcl 2 domain protected brain structure from extensive damage. Therefore, Mentha extract seems to have a significant role to ameliorate the neuronal injury induced by gamma irradiation. Content Type Journal Article Category Original Research Pages 1-12 DOI 10.1007/s10616-012-9470-1 Authors Hanaa A. Hassan, Physiology Division, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt Hani S. Hafez, Zoology Department, Faculty of Science, Siez, Suez Canal University, Ismailia, Egypt Mona S. Goda, Genetic Department, Hospital Medicine, Mansoura University, Mansoura, Egypt Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg −1 and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”. Content Type Journal Article Category Method in Cell Science Pages 1-16 DOI 10.1007/s10616-012-9491-9 Authors P. Jayesh, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India Seena Jose, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India Rosamma Philip, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India I. S. Bright Singh, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Polyethylenimine (PEI) has been used widely in transient gene expression studies of mammalian cells. We performed transient gene expression in suspension Chinese hamster ovary cells using a one-step transfection procedure in which DNA and PEI were simultaneously added to a cell culture in suspension without prior PEI/DNA complex incubation. To further understand the effect of PEI/DNA formation on the transfection and expression of exogenous gene in shaking state, we investigated the diameter and overcharge of the PEI/DNA complex. The results showed that the diameter of the complex was smaller with more positive charge when the PEI/DNA ratio was higher. Moreover, DNA more easily penetrated cells and nuclei at higher PEI concentrations. The highest transcription level, transfection efficiency, and GFP expression were obtained when the PEI/DNA ratio was 5:1. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9483-9 Authors Qiuling Xie, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Guo Xinyong, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Chen Xianjin, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Wang Yayu, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Pdx - 1 and Irs - 1 , genes highly associated with diabetes onset, were knocked down in mouse embryonic stem (ES) cells in order to develop cell line models for diabetes. ES cells with different gene knockdown levels were induced to differentiate to the stage of insulin production. Among the cell lines that differentiated, we identified two in which the levels of expression of both genes were 20–40 % of that of control cells. These cell lines showed appreciable deficiencies in three characteristic malfunctions associated with diabetes, namely, insulin production, insulin reception signaling, and glucose-stimulated insulin secretion. These dysfunctions were consistent with results reported elsewhere from in vivo and in vitro studies. Both cell lines did not show any abnormal morphology such as size, shape, color, and surface roughness. No abnormal expression profiles for 17 genes relevant to diabetes were observed. Therefore, these cell lines fulfilled the criteria for a validated cell model for diabetes. The model cell lines developed here are promising biomaterials for cell-based screening tests of new medicines that may be effective in treating diabetes. Content Type Journal Article Category Original Research Pages 1-14 DOI 10.1007/s10616-012-9466-x Authors Mikako Saito, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588 Japan Aya Hayakawa, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588 Japan Nobuya Inagaki, Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507 Japan Hideaki Matsuoka, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Open reading frame 17 ( Bm17 ) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9451-4 Authors Hongxing Shen, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Yang Zhou, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Wen Zhang, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Bin Nin, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Hua Wang, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Xiaochun Wang, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Shihe Shao, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Huiqing Chen, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Zhongjian Guo, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Xiaoyong Liu, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Qin Yao, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Keping Chen, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H 2 O 2 levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H 2 O 2 level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC 50 ) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC 50 by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H 2 O 2 levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening. Content Type Journal Article Category Original Research Pages 1-7 DOI 10.1007/s10616-011-9423-0 Authors Wenwei Mao, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Xinlin Chen, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Tian Yang, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Yu Yin, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Mei Ge, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Minyu Luo, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Daijie Chen, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Xiuping Qian, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069