Radiation-inducible TNF-α gene expression under stress-inducible promoter gadd 153 for cancer therapy

We demonstrated the effectiveness of radiation-inducible expression of the TNF-α gene for cancer therapy in vitro. The TNF-α gene under the control of the stress-inducible promoter, gadd 153, was introduced into the human glioma cell line, U251-SP. Without cobalt-60 gamma irradiation, no cytotoxicity against the transfected cells was observed. When the transfected cells were irradiated with 10 or 20 gray (Gy), the gadd 153 promoter was highly induced and the expression level of TNF-α increased. Five days after the irradiation, the TNF-α productions of each cell irradiated with 10 and 20 Gy were 30 and 100 times higher than the basal level, respectively. The cytotoxicities against the transfected cells 5 d after irradiation with 10 and 20 Gy were 79% or 91%, respectively, which are much higher than those against the nontransfected cells that were irradiated at the same dose (43% and 78%, respectively). These results demonstrate that the gadd 153-TNF-α system may be an effective tool for radiosurgery of malignant brain tumors.     

Bystander-killing effect and cyclic induction of TNF-alpha gene under heat-inducible promoter gadd 153

The bystander-killing effect and cyclic induction of tumor necrosis factor (TNF)-alpha gene expression under the heat-inducible promoter gadd 153 (growth arrest and DNA damage) were investigated. The plasmid harboring the TNF-alpha gene under the control of the gadd 153 promoter (pGadTNF) was introduced into cells of the human glioma cell line U251-SP. When the transfected cells were exposed to heat treatment, TNF-alpha gene expression was induced. The heated pGadTNF-transfected cells were co-cultured the nonheated pGadTNF-transfected cells as bystander cells. As a result, TNF-alpha gene expression in the bystander cells was observed. The level of TNF-alpha gene expression was further enhanced in the co-cultured cells. Due to this cyclic induction, a strong cytotoxic effect on the bystander cells was observed. Based on this bystander-killing effect, it was concluded that the transfection of the TNF-alpha gene under the control of the gadd 153 promoter into tumor cells combined with hyperthermia would be a potent tool for cancer therapy.     

Cystone,an ayurvedic herbal drug imparts protection to the mice against the lethal effects of gamma-radiation: a preliminary study

The effect of 5, 10, 20, 40, 60, 80, 100, 120, and 160 mg/kg body weight (b.wt.) of aqueous extract of cystone (an ayurvedic herbal medicine) administered intraperitoneally was studied on the radiation-induced mortality in mice exposed to 10 Gy of gamma-radiation. Treatment of mice with different doses of cystone, consecutively for five days before irradiation, delayed the onset of mortality and reduced the symptoms of radiation sickness when compared with the non-drug treated irradiated controls. The pretreatment of mice with different doses of cystone before exposure to 10 Gy of gamma-radiation resulted in a dose-dependent elevation in the survival up to 40 mg/kg b.wt., where the highest number of survival (55.55%) was observed by 30 days post irradiation, when compared with the 10 Gy irradiated control (6.66%). Thereafter, the number of survivors declined and reached a nadir at 160 mg/kg, where no survivors could be observed. The optimum protection against irradiation was observed for 40 mg/kg cystone, where the highest number of survivors were reported by 30 days post irradiation and it was 8.34-fold greater than that of the irradiated control group.     

Cystone,an ayurvedic herbal drug imparts protection to the mice against the lethal effects of γ‐radiation: A preliminary study

The effect of 5, 10, 20, 40, 60, 80, 100, 120, and 160 mg/kg body weight (b.wt.) of aqueous extract of cystone (an ayurvedic herbal medicine) administered intraperitoneally was studied on the radiation‐induced mortality in mice exposed to 10 Gy of γ‐radiation. Treatment of mice with different doses of cystone, consecutively for five days before irradiation, delayed the onset of mortality and reduced the symptoms of radiation sickness when compared with the non‐drug treated irradiated controls. The pretreatment of mice with different doses of cystone before exposure to 10 Gy of γ‐radiation resulted in a dose‐dependent elevation in the survival up to 40 mg/kg b. wt., where the highest number of survival (55.55%) was observed by 30 days post irradiation, when compared with the 10 Gy irradiated control (6.66%). Thereafter, the number of survivors declined and reached a nadir at 160 mg/kg, where no survivors could be observed. The optimum protection against irradiation was observed for 40 mg/kg cystone, where the highest number of survivors were reported by 30 days post irradiation and it was 8.34‐fold greater than that of the irradiated control group.     

Effects of gamma irradiation on fourth-instar Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae)

Effects of gamma irradiation on fourth-instar Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) larvae in infested dates (Boufeggous variety) were assessed. Larvae were exposed to different gamma irradiation doses ranging from 300 to 900 Gy. Feeding, pupation, adult emergence and survival were very sensitive to ionizing irradiation. When irradiated at a dose of 300 Gy and higher, food consumption and weight gain were significantly affected, in a dose-dependent manner. Twenty days after irradiation, the weight loss at doses of 300, 450 and 600 Gy was 42%, 47% and 49%, respectively. At doses of 750 and 900 Gy, the weight of larvae diminished by 51% and 54%, respectively. In contrast, the controls gained 20% in weight. Development of larvae to the pupal stage was not prevented completely but none of the pupae emerged as adults. At 300 Gy, 21% of larvae survived 44 d after irradiation and successfully pupated. An absorbed dose of 450, 600, 750 or 900 Gy applied to larvae caused 100% mortality by 44, 38, 38 and 22 d post-treatment, respectively. The minimum dose required to prevent damage from feeding and adult emergence was 300 Gy while doses required to cause death were ?450 Gy.     

Effects of Gamma Irradiation Dose and Timing of Treatment after Harvest on the Storeability of Garlic Bulbs

The effect of gamma irradiation dose and time of treatment after harvest on the storage of garlic bulbs was investigated. The effectiveness of irradiation for external sprout inhibition was not affected by the treatment time within 45 days after harvest. At 285 days after harvest, irradiation of 50 - 150 Gy caused about 6% less decrease in weight loss compared with the unirradiated group, and spoilage rates of the unirradiated and irradiated cloves were 100% and 17 - 20%, respectively. For the overall storageability of garlic bulbs, 75 Gy was shown to be the minimal optimum dose, and there was no apparent effect depending upon the time of irradiation treatment after harvest.     

Autologous and homologous transplantation of bovine spermatogonial stem cells

The aim of this study was to develop a method for spermatogonial stem cell transplantation into the bovine testis. Five-month-old Holstein-Friesian calves were used and half of the calves were hemicastrated to allow autologous transplantation and the other half were used for homologous transplantation. Approximately 20 g of each testis was used for cell isolation. On average 106 cells per gram of testis containing about 70% type A spermatogonia were isolated. The cells were frozen in liquid nitrogen until transplantation. Testes were irradiated locally with 10-14 Gy of X-rays to deplete endogenous spermatogenesis. At 2 months after irradiation, cells (approximately 10 x 10(6) were injected into the rete testis through a long injection needle (18 gauge), using ultrasonography and an ultrasound contrast solution. At 2.5 months after transplantation, calves were castrated and samples of testes were taken for histological examination. After 2.5 months in the irradiated non-transplanted control testes, only 45% of the tubules contained type A spermatogonia. However, after autologous spermatogonial transplantation, >80% of the tubule cross-sections contained type A spermatogonia. In addition, only 20% of the tubules of the control testes contained spermatocytes and, except for a few tubules (5%) with round spermatids, no more advanced germ cells were found. After autologous spermatogonial transplantation, about 60% of the tubules contained spermatocytes; 30% contained spermatids and in about 15% of tubules spermatozoa were found. No improvement in spermatogonial repopulation was found after homologous transplantation. The results of this study demonstrate, for the first time, successful autologous transplantation of bovine spermatogonial stem cells resulting in a complete regeneration of spermatogenesis.     

Evaluation of the radioprotective effect of the leaf extract of Syzygium cumini (Jamun) in mice exposed to a lethal dose of γ‐irradiation

The effects of various concentrations (5, 10, 20, 30, 40, 50, 60, and 80 mg/kg body weight (b.wt.) of the leaf extracts of Syzygium cumini Linn. and Eugenia cumini (SCE, black plum, Jamun, family Myrtaceae) on the radiation‐induced sickness and mortality in mice exposed to 10 Gy γ‐irradiation were studied. The treatment of mice with different doses of SCE, consecutively for five days before irradation, delayed the onset of mortality and reduced the symptoms of radiation sickness when compared with the nondrug‐treated irradiated controls. All doses of SCE provied protection against the gastrointestinal death increasing the survival by 66.66% after treatment with 20, 30, and 40 mg/kg SCE versus a 12% survival in the irradiated control group (oil + irradiation). Similarly, SCE provided protection against the radiation‐induced bone marrow death in mice treated with 10–60 mg/kg b.wt. of SCE. However, the best protection was obtained for 30 mg/kg b.wt. SCE, where the number of survivors after 30 days post‐irradiation was highest (41.66%) when compared with the other doses of SCE.     

Increase in gene expression by respiratory-deficient mutation

Respiratory-deficient mutants (rho- cells) of Saccharomyces cerevisiae produced about 10 times as much human (h-) lysozyme as did wild-type strains (rho+ cells) when the GAL10 promoter was used in an expression plasmid with the h-lysozyme gene. Introduction of intact mitochondria into the rho- cells resulted in a significant decrease in the production of h-lysozyme, indicating that the rho- mutation increased the expression of the h-lysozyme gene. The copy number of the expression plasmid was not responsible for the increased expression. The level of h-lysozyme mRNA in the rho- cells was also much higher than that in the rho+ cells especially at the stationary phase. The increased expression of the h-lysozyme gene was also observed when a glyceraldehyde-3-phosphate dehydrogenase gene promoter and the PHO5 promoter were used in the expression plasmid. The rho- mutation also increased the expression of the PHO5 gene under the control of the HIS5 promoter in a plasmid and the ACT1 gene in the yeast chromosome, but did not increase the expression of the ribosomal RNA gene. In contrast to the rho- mutants, pet mutants did not show higher gene expression compared with wild-type strains.     

Evaluation of the radioprotective effect of the leaf extract of Syzygium cumini (Jamun) in mice exposed to a lethal dose of gamma-irradiation

The effects of various concentrations (5, 10, 20, 30, 40, 50, 60, and 80 mg/kg body weight (b.wt.) of the leaf extracts of Syzygium cumini Linn. and Eugenia cumini (SCE, black plum, Jamun, family Myrtaceae) on the radiation-induced sickness and mortality in mice exposed to 10 Gy gamma-irradiation were studied. The treatment of mice with different doses of SCE, consecutively for five days before irradation, delayed the onset of mortality and reduced the symptoms of radiation sickness when compared with the nondrug-treated irradiated controls. All doses of SCE provied protection against the gastrointestinal death increasing the survival by 66.66% after treatment with 20, 30, and 40 mg/kg SCE versus a 12% survival in the irradiated control group (oil + irradiation). Similarly, SCE provided protection against the radiation-induced bone marrow death in mice treated with 10-60 mg/kg b.wt. of SCE. However, the best protection was obtained for 30 mg/kg b.wt. SCE, where the number of, survivors after 30 days post-irradiation was highest (41.66%) when compared with the other doses of SCE.     

摄入酸奶对辐照小鼠体重和免疫细胞的影响

辐射前后5d用含保加利亚乳杆菌和嗜热链球菌活菌的酸奶添加于饲料中饲喂小鼠，比较接受2Gy辐射小鼠的体重、胸腺细胞重量、腹膜巨噬细胞吞噬活性和脾淋巴细胞计数等指标，发现摄入添加酸奶饲料的处理小鼠与普通饲料对照组有显著的差异，证明饲喂酸奶对辐射后小鼠产生了保护作用，减轻其体重下降、胸腺重下降和淋巴细胞计数，提高其巨噬细胞吞噬活性。     

高能电子束辐照对猕猴桃细胞壁降解相关酶活性和基因表达的影响

以‘海沃德’猕猴桃为试材,经剂量0（对照）、300、400和500 Gy高能电子束辐照后,于0~1 ℃、RH 90%~95%冷库中贮藏90 d,研究电子束辐照对果实硬度、细胞壁组分、软化相关酶活性及其基因表达量的影响。结果表明:高能电子束辐照显著维持了果实的硬度,有效抑制了细胞壁骨架物质原果胶和纤维素的分解,延迟了果实后熟软化。同时,辐照抑制了多聚半乳糖醛酸酶（polygalacturonase,PG）、果胶甲酯酶（pectin methylesterase,PME）、β-半乳糖苷酶（β-D-galaetosidase,β-Gal）和纤维素酶（cellulase,Cx）的活性,降低了PG、PME、β-Gal和Cx编码基因的表达。综合认为,以400 Gy高能电子束辐照对抑制细胞壁降解相关酶活性及基因表达,保持细胞结构的完整性,维持贮藏期间果实硬度效果最好。研究结果为高能电子束用于猕猴桃采后保鲜提供理论依据。     

高能电子束与60Coγ射线对大蒜辐照保鲜效果的比较研究

我国大蒜辐照抑芽工艺规范没有对60Coγ射线和电子束应用的工艺参数做出区分,为明确两者对大蒜抑芽保鲜效果的差异,以60Co 产生的γ射线和电子加速器产生的高能电子束,分别对大蒜进行辐照处理,辐照剂量为200、500、800Gy,辐照后置于室温(5～25℃,RH 70%～85%)贮藏,并对贮藏期间大蒜内芽生长情况及各生理指标进行测定。结果表明：两种辐照源均能有效抑芽,且相同辐照剂量下,电子束辐照抑芽效果强于60Coγ射线;200Gy 和500Gy 的两种辐照均能抑制大蒜呼吸作用,延缓质量损失,且对大蒜鳞茎外皮颜色和大蒜风味品质影响不大;而高剂量800Gy 的两种辐照则刺激大蒜呼吸增强,失重率升高,大蒜干缩严重。从大蒜营养品质和商业品质的角度综合分析,电子束采用200Gy、60Coγ射线采用500Gy 对大蒜的抑芽保鲜效果较好。     

Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5′-flanking regions of the human p21 gene

A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy γ-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-κB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.     

小鼠PKM1基因慢病毒过表达载体的构建和稳定转染C2C12细胞株的筛选及其对糖酵解的影响

本研究旨在构建含小鼠PKM1基因的慢病毒过表达载体,筛选稳定过表达PKM1的C₂C₁₂细胞株,初步检测PKM1对细胞糖酵解的影响。提取小鼠背最长肌总RNA并反转录合成cDNA。用"Golden Gate"法构建带FLAG标签的PKM1表达载体并转染293T细胞,进行慢病毒包装并检测慢病毒滴度。转染C₂C₁₂细胞并优化转染条件,根据载体携带的抗性基因puro,用嘌呤霉素筛选稳转株。通过qPCR和Western Blot从转录水平和翻译水平检测目的基因的表达。通过测定细胞培养基的pH、乳酸含量以及葡萄消耗量检测PKM1对糖酵解的影响。结果表明小鼠PKM1基因慢病毒载体构建成功,慢病毒滴度为3.4×108 TU/mL。慢病毒侵染靶细胞的最佳感染复数为30,筛选稳转细胞株所用嘌呤霉素的最佳浓度为1.2 μg/mL。显微镜下观察病毒转导效果良好,荧光率达80%以上。过表达组中FLAG-PKM1的mRNA表达量约是空载体组的9.4万倍。FLAG-PKM1融合蛋白成功在宿主细胞内表达。与野生型组和空载体组相比,过表达组细胞培养基的乳酸含量增加,葡萄糖消耗量增加,pH降低,表明细胞糖酵解加剧。本研究成功构建了含小鼠PKM1基因的稳转株,为深入研究PKM1翻译后修饰对其自身酶活以及细胞糖酵解的影响提供了材料。     

木犀草素与叶酸对黄曲霉毒素B1致食管上皮细胞毒性及MTHFR基因高甲基化的影响

目的：研究木犀草素（luteolin,LUT）与叶酸（folic acid,FA）对黄曲霉毒素B1（aflatoxin B1,AFB1）诱导损伤的人正常食管上皮细胞（human normal esophageal epithelial cells,HEEC）MTHFR基因甲基化的影响。方法：不同浓度（0、13、25、50、100、200 μmol/L）AFB1染毒HEEC 24、48、72 h,CCK-8法检测细胞活力;将HEEC分为空白对照组、AFB1染毒组（200 μmol/L）、LUT干预组（160 μmol/L）、FA干预组（20、200 μmol/L）以及联合干预组（160 μmol/L LUT＋20 μmol/L FA、160 μmol/L LUT＋200 μmol/L FA）,处理24 h后CCK-8法检测细胞活力,流式细胞术检测细胞周期及凋亡,Western blot检测MTHFR蛋白的表达水平,MassARRAY甲基化检测MTHFR基因启动子区甲基化的情况。结果：不同浓度的AFB1染毒HEEC 24、48、72 h均可以抑制细胞增殖,而经LUT和FA干预后,与AFB1染毒组相比,LUT及联合干预组细胞周期阻滞显著减少（P＜0.05）,细胞抑制率及凋亡率显著降低（P＜0.05）,MTHFR蛋白表达上调有所改善（P＜0.05）。且LUT与FA及二者联合对MTHFR基因启动子区高甲基化水平均有降低作用（P＜0.05）。结论：AFB1对HEEC有毒性作用,表现在增殖抑制、周期阻滞,促进凋亡,上调了MTHFR蛋白的表达,LUT干预可减弱这些损伤,对AFB1所致毒性起到一定的保护作用。AFB1提高了MTHFR基因启动子区甲基化水平,导致表观遗传的改变。LUT与FA及联合作用可以降低该甲基化水平,改善该表观遗传的改变。     

Radiation resistance and virulence of Listeria monocytogenes Scott A following starvation in physiological saline

The influence of starvation on the resistance of Listeria monocytogenes Scott A to electron beam irradiation in 0.85% (wt/vol) NaCl (saline) and in ground pork was investigated. Exponential- or stationary-phase cells (control) were grown at 35 degrees C in tryptic soy broth supplemented with 0.6% yeast extract. Washed cells were starved for 12 days in saline, and virulence of the pathogen was evaluated at 0, 8, and 12 days during starvation. Samples of saline and irradiation-sterilized ground pork, inoculated with control or starved cells, were irradiated at doses ranging from 0.0 to 2.5 kGy. L. monocytogenes survivors were determined by plating diluted samples of saline or pork on tryptic soy agar supplemented with 0.6% yeast extract and counting bacterial colonies following incubation (35 degrees C, 48 h). Virulence of starved cells and control was not significantly different (P > 0.05). Cells exhibited the highest radiation resistance at 8 days of starvation. Irradiation (0.5 kGy) in saline resulted in approximately 7.14, 5.55, and 2.38 log reduction in exponential, stationary, and starved cells, respectively. Irradiation of ground pork at 2.5 kGy reduced controls by approximately 6.0 log, whereas starved cells were reduced by only 3.8 log. Starved cells consistently exhibited higher irradiation D10-values than controls (P < 0.05). D10-values for exponential, stationary, and starved cells were 0.07, 0.09, and 0.21 kGy and 0.35, 0.42, and 0.66 kGy in saline and ground pork, respectively. These results indicate that starvation cross-protects L. monocytogenes Scott A against radiation inactivation and should be considered when determining this pathogen's irradiation D-value.     

Antioxidant potential and radioprotective effect of soy isoflavone against gamma irradiation induced oxidative stress

The in vitro antioxidant potential and in vivo radioprotective ability of soy isoflavones was studied. Male Wistar rats were orally administered with soybean isoflavones (60 mg/kg) for 21 days followed by gamma irradiation exposure. Survival studies in rats exposed at 10 Gy and endogenous spleen colony forming unit assay (CFU) at 6.0 Gy were performed in order to find radioprotective and immunomodulatory nature of the compound. The rat liver post mitochondrial supernatant and erythrocytes were used to measure lipid peroxidation (LPO) and glutathione (GSH) content along with various antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) after gamma irradiation exposure at 2.0 Gy. Pretreatment with soy isoflavone, prior to gamma irradiation resulted in the increased survival rate of the animals as compared to irradiated group. CFU counts in the isoflavone treated group followed by gamma irradiation at 6 Gy were significantly high as compared to control and the irradiated group, showing immunomodulatory nature of the isoflavones. Pretreatment with isoflavones also significantly reduced the LPO, enhanced the activity of antioxidant enzymes and improved haematological and histological parameters. The present study suggests that supplementation with isoflavone has potent antioxidant activity and act as probable radioprotector against gamma radiation induced oxidative damage.     

Low‐Dose Irradiation Can be Used as a Phytosanitary Treatment for Fresh Table Grapes

Grapes (Vitis vinifera var. Sugraone and Vitis labrusca var. Crimson Seedless) were treated with 400, 600, and 800 Gy and the effects on physicochemical factors were measured alongside sensory testing during 3 wk of storage. Significant changes in texture and color with irradiation and age were measured but little visual difference was seen between control and irradiated grapes. However, age had a greater effect on firmness than irradiation for Sugraone grapes. Irradiation did not significantly (P ≤ 0.05) affect the SSC/TA ratio, which increased during storage. The trained panel detected significant changes in the berry texture and rachis color but rated sweetness and flavor significantly higher (P ≤ 0.05) for irradiated Sugraone as compared to the control. Consumers liked both the untreated and 800 Gy treated Sugraone grapes, but liked the untreated grapes more for texture (P ≤ 0.05). However, there was no difference in liking between irradiated (600 Gy or 800 Gy) and control samples of Crimson Seedless for any attribute. The results show that there are varietal differences in response to irradiation but the overall maintenance in quality of irradiated grapes during 3 wk of storage indicates that irradiation can serve as a viable phytosanitary treatment.