Construction of DNA ladder based on 16S rRNA gene of Bacillus subtilis using touchdown PCR technique

Aim@#DNA molecular size markers or DNA ladders play a vital role in molecular biology laboratories where DNA electrophoresis experiments are usually conducted. This study aimed to produce a 100 bp DNA ladder at laboratory scale, which could be applied to determine the size of DNA fragments in molecular biology experiments.@*Methodology and results@#In this study, 14 primers including 4 forwards and 10 reverses were designed based on the 16S rRNA gene sequence of Bacillus subtilis. These primers were able to amplify 10 DNA fragments with accurate sizes from 100 to 1000 bp. Furthermore, touchdown PCR was involved to maximize the specificity and yield of PCR products. Ten DNA fragments with the sizes including 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 bp were synthesized, and such bands were equivalent with commercial DNA ladders. Moreover, the quantity and quality of PCR products were measured using a nanodrop spectrophotometer. The optimal concentration ratios between such fragments (100- 1000 bp) were 800, 300, 150, 150, 500, 50, 50, 50, 50 and 50 (ng/µL), respectively. These ratios showed the clear and high resolution on 1.5% agarose gel. @*Conclusion, significance and impact of the study@#The results indicated that 16S rRNA gene of B. subtilis was a potential material for DNA ladder preparation due to the multiple copies number of this gene. Furthermore, in combination with touchdown PCR, the nonspecific bands were reduced, and the products could be used directly without the need of purification step.

Protective action against chemical-induced genotoxicity and free radical scavenging activities of Stryphnodendron adstringens ("barbatimão") leaf extracts

The present study describes for the first time the antigenotoxic and antioxidant properties of Stryphnodendron adstringens (Mart.) Coville, Fabaceae ("barbatimão") leaves. The aqueous (AEB), water fraction (WFB) and ethanolic (EEB) extracts of leaves were obtained and they presented high content of phenolic compounds, flavonoids and proanthocyanidins. WFB and EEB inhibited the genotoxicity induced by cyclophosphamide. WFB inhibited the DNA lesion formation and both WFB and EEB decreased significantly (p<0.05) micronucleus formation. All extracts also showed high DPPH radical scavenging activity and reducing power. In conclusion all extracts presented antioxidant activity and WFB and EEB protected cells from genotoxicity induced by cyclophosphamide in rat bone marrow cells. Thus, our results support the beneficial effects of the barbatimão extracts as an anticarcinogenic agent.

The damaging effect of sulfur mustard on the mitochondria in rat lymphocytes in vitro / 第三军医大学学报

Objective To investigate the damaging effect of sulfur mustard on the mitochondria in rat lymphocytes in vitro.Methods Rat spleen lymphocytes were isolated by density gradient centrifugation and cultured with 100 ?mol/L sulfur mustard in vitro.DNA ladderring was used to detect the cell apoptosis.Cyt-c release was measured by Western blotting.The mitochondria function was detected by MTT method.Fluorescent probe labeled with Rhodamine 123 was used to study mitochondria potential.Results The mitochondria potential and cell viability decreased at 4 h after treated with 100 ?mol/L sulfur mustard.There was a liner correlation between the mitochondria potential and cell viability in lymphocytes.The content of Cyt-c increased significantly at 4 h as compared with control group in Western blotting.Conclusion Sulfur mustard could damage the rat lymphocyte mitochondria significantly,and mitochondria participates in cytotoxic effect induced by the sulfur mustard.

Studies on the Radiation Induced Apoptosis by Morphological and Biochemical Analysis in A431 Cells / 대한핵의학회잡지

PURPOSE: We performed this study to evaluate the process of radiation induced apoptosis in A431 skin epithelial cancer cell line. MATERIALS AND METHODS: Low to high dose radiation (0, 2, 5, 10, 25 Gy) was given to A431 cells by Cs-137 cell irradiator. Apoptosis was evaluated by cell morphology, dye exclusion test, and DNA laddering. RESULTS: Cell viability decreased as the radiation dose increased. Number of apoptotic bodies increased as radiation dose increased. It increased most significantly at 12 hours after irradiation. Lactate dehydrogenase activity in culture medium increased according to radiation dose and time after irradiation. CONCLUSION:: Radiation-induced apoptosis which was the main course of cell death in A431 cells could be analyzed quantitatively by counting apoptotic bodies under microscope. Apoptosis increased as radiation dose increased.

Nickel (II)-induced apoptosis and G2/M enrichment

Treatment with certain DNA-damaging agents induce a complex cellular response comprising pertubation of cell cycle progression and/or apoptosis on proliferating mammalian cells. Our studies were focused on the cellular effects of nickel (II) acetate, DNA-damaging agent, on Chinese hamster ovary (CHO) cells. Fragmented DNAs were examined by agarose gel electrophoresis and cell cycle was determined by DNA flow cytometry using propidium iodide fluorescence. Apparent DNA laddering was observed in cells treated with 240 microM nickel (II) and increased with a concentration-dependent manner. Treatment of nickel (II) acetate resulted in apoptosis which was accompanied by G2/M cell accumulation. Proportion of CHO cells in G2/M phase was also significantly increased in cells exposed to at least 480 microM nickel (II) from 57.7% of cells in the G0/G1 phase, 34.7% in the S phase, and 7.6% in the G2/M1 phase for 0 microM nickel (II), to 58.6%, 14.5%, and 26.9% for 640 microM nickel (II). These findings suggest that nickel (II) can modulate cellular response through some common effectors involving in both apoptotic and cell cycle regulatory pathways.

Antagonizing effects of GSH against rat lymphocytotoxicity induced by sulfur mustard / 第三军医大学学报

Objective To investigate the protective effects of GSH on the mitochondria in rat lymphocyte in vitro.Methods Rat spleen lymphocytes were isolated by density gradient centrifugation and cultured with sulfur mustard of 100,500,1000 ?mol/L in vitro.The mitochondria function was detected by MTT.The changes of GSH were detected by fluorescence spectrophotometry and DNA fragments were also detected.Results There was a positive line correlation between the changes of mitochondria function and the GSH content(P