酵母菌小菌落突变株酒精发酵的研究——Ⅰ.菌种的选育及其生理特性

酵母菌小菌落呼吸缺陷型突变株(Petite mutant)的研究国外已有许多报道。Bacila等证明ρ~-突变株在5L发酵罐中酒精发酵产量比野生型ρ~+约提高10%左右。我们用诱变剂对酒精工业上使用的酵母菌进行处理,得到呼吸缺陷型突变株,这些菌与氯     

溴化乙锭诱导无线粒体DNA宫颈癌HeLa细胞系的构建和鉴定

[目的]构建和鉴定由溴化乙锭(EB)诱导的无线粒体DNA(mtDNA)宫颈癌ρ~0HeLa细胞系,探讨mtDNA与宫颈癌发生的关系。[方法]采用含50ng/ml溴化乙锭、100μg/ml丙酮酸钠和50μg/ml尿嘧啶核苷的高糖DMEM完全培养基中传代培养HeLa细胞。低剂量EB连续诱导60d后,采用营养缺陷鉴定、PCR和WesternBlot鉴定无mtDNA的ρ~0HeLa细胞系;采用透射电子显微镜观察ρ~0HeLa细胞内线粒体形态变化;采用CCK8法测定ρ~0HeLa细胞增殖曲线。[结果]经溴化乙锭诱导60d,可以培养出具有尿嘧啶核苷依赖性的无mtDNA宫颈癌HeLa细胞系。普通PCR和qPCR结果均显示,低剂量EB诱导60d的ρ~0HeLa细胞中mtDNA完全缺失。WesternBlot结果显示,HeLa细胞中能表达核编码的NDUFA9蛋白,也能表达线粒体编码MT-ND1蛋白。而ρ~0HeLa细胞中已无MT-ND1蛋白表达,但核编码的NDUFA9蛋白能够正常表达。透射电子显微镜观察显示,ρ~0HeLa细胞内部分出现空泡改变,线粒体嵴被破坏。CCK8细胞增殖实验结果显示,ρ~0HeLa细胞系生长速度显著低于正常HeLa细胞系,且差异有统计学意义(P<0.05)。[结论]无mtDNA的宫颈癌HeLa细胞系的建立,为后续研究mtDNA突变和线粒体功能在宫颈癌发生发展中的作用及机制奠定了基础。     

人线粒体DNA缺失肝癌细胞株建立

人线粒体DNA缺失肝癌细胞株(ρ0SK-Hep1)的建立及鉴定。采用溴化乙锭(EB)诱导,PCR、Southern杂交及选择性培养方法进行鉴定。ρ0SK-Hep1细胞在含EB、无尿嘧啶和丙酮酸的选择培养基中从第1天始细胞逐渐悬浮、肿胀,培养第5天以后大量悬浮死亡,且贴壁疏松,10～12天细胞完全悬浮死亡。在选择性培养基中可以正常生长增殖成单层,有少量悬浮。同期非选择培养的SK-Hep1细胞生长正常。PCR结果显示,细胞色素氧化酶I、II(COXI、COXII)及内参G3PDH在SK-Hep1细胞中均可扩增出相应的条带,ρ0SK-Hep1细胞只见内参条带的形成。Southern杂交结果显示,ρ0SK-Hep1细胞未见COXI、COXII杂交条带形成。经EB诱导后成功地获得了ρ0SK-Hep1细胞株。     

无线粒体DNA的人肺腺癌A549细胞系的构建

目的:建立无线粒体DNA(mtDNA)的人肺腺癌ρ~0A549细胞系。方法:在含50 ng/mL溴化乙锭(EB)、100μg/mL丙酮酸钠和50μg/mL尿嘧啶核苷的RPMI1640细胞培养基中传代培养A549细胞;用低剂量EB连续诱导培养35 d后,采用光镜观察、TaqMan探针法实时荧光定量PCR(qPCR)和Western印迹鉴定无mtDNA的ρ~0A549细胞系;采用MTT法测定ρ~0A549细胞增殖曲线。结果:倒置显微镜下野生型ρ^+A549细胞为多角形,ρ~0A549细胞形态呈拉长枝状;qPCR结果显示,低剂量EB诱导35 d的ρ~0A549细胞中无mtDNA的存在。Western印迹结果显示,ρ^+A549细胞中能表达核基因编码的线粒体蛋白SDHA和ATP5A,也能表达线粒体基因组编码的蛋白MT-COXI和MT-ATP6;ρ~0A549细胞中无MT-COXI和MT-ATP6蛋白表达,但核基因编码的SDHA和ATP5A蛋白能够正常表达。MTT结果显示,与ρ^+A549细胞相比,ρ~0A549细胞生长速度明显减慢,差异有统计学意义(P<0.05)。结论:构建和鉴定了无mtDNA的人肺腺癌ρ~0A549细胞系,为后续探讨mtDNA缺失或突变与人肺腺癌发生之间的关系奠定了实验基础。     

N-甲基-N'-硝基－N－亚硝基孤对阴沟肠杆菌诱变作用的研究

营养缺陷型无论在微生物遗传学的研究或 诱变育种方面皆有着广泛的用途。由于亚硝基 孤(NTG）是一种诱变作用特别强的诱变剂, 所以应用也较为广泛,对于细菌的诱变作用的 研究已有不少报道［[1181,而对阴沟肠杆菌的诱变 作用尚未见到。本文以该菌为材料,对NTG 诱变剂的使用浓度与处理时间的选择,及应用 青霉素浓缩法与上述两因素的联合作用,以及 三者之间的关系作了研究。我们采用3个因素 （诱变剂浓度、处理时间、浓缩法的青霉素浓 度）,4个水平,16个组合的正交设计[21,研究 了最大限度地获得营养缺陷型的最适条件,对 所获得的营养缺陷型进行了鉴定,对其固氮活 性进行了分析,结果报道如下。     

氮素营养对红萍生理特性的影响 Ⅰ硝态氮的作用

不同氮源对红萍(Azolla imbricata)的生长和固氮作用都有影响。尿素的抑制作用最为严重,铵态氮次之,硝酸态氮最轻,硝酸铵作用介于铵态氮和硝酸态氮之间。这几种氮源对萍体内游离氨基酸的组分和含量也有影响,变化较大的为苏氨酸和精氨酸。在0.05～51.2 mM NO_3~-浓度范围内,红萍的固氮活性随着NO_3~-浓度变化呈双抛物线形变化,在高浓度下,活性急剧下降。硝酸还原酶活性则相反,与固氮活性相互补。应用~(15)NO_3~-实验结果表明,在红萍体内~(15)NO_3~-能迅速还原成~(15)NH_4~+。NO_3~-浓度对萍体内叶绿素含量和a/b比影响不大。     

张士灌区土壤中镉形态的探讨

本文研究了张士灌区土壤中镉的形态。交换态占78％,可给态占18％,不溶态和碱溶态只占3—4％。在田间酸性土壤中,Cl~-是无机配位体的主要成分。镉主要与Cl~-络合而形成[CdCl]~-和CdCl_2。土壤中加入石灰和钙、镁、磷肥、HCO_3~-增多,镉与之生成Cd(HCO_3)_2沉淀。在长期淹水状况下,S~-增多,镉与S~-产生CdS沉淀,难以被水稻吸收。     

莫桑鼻给非鲫滤泡闭锁中液晶形成的光镜和电镜观察

许多研究结果表明生物体內普遍存在流动而有序的液晶态结构,液晶态和生命活动的关系已受到人们的高度重视。在研究莫桑鼻给非鲫的卵子发生时发现,卵巢内存在大量的滤泡闭锁现象。同时发现这种现象伴随着液晶态的出现。滤泡闭锁和液晶形成的关系如何?液晶形成的意义何在?为此,运用光镜和电镜观察了滤泡闭锁的过程。     

聚磷菌的诱变选育及其生长特性

方法:采用氮离子注入技术对巢湖底泥中筛选出的一株细菌进行辐照诱变处理,选育出2株高效聚磷菌,并对这两株菌的生长特性进行了研究,根据生理生化及生长试验,并参照《伯杰氏细菌手册》进行菌种分类鉴定,这两株菌被鉴定为假单胞菌属细菌(Pseudomonas sp),并研究了菌量、温度、pH、氧、碳源对两株假单胞菌(Pseudomonas sp)的生长特性及聚磷代谢的影响。结果:试验表明,诱变后的聚磷率明显高于试验出发菌,为出发菌的1.43~3.89倍。两株菌的最适生长温度为30℃,最适pH和菌量分别为6、8和OD=0.6、0.4。最适菌量在厌氧条件下出现放磷现象,好氧条件下过量摄磷现象,经过先厌氧条件的培养其去磷率明显高于直接好氧培养条件下的去磷率,以乙酸钠为碳源时其聚磷率高于乳糖、甲醇、乙醇为碳源时的摄磷量。     

卡那霉素链霉菌亚硝基胍诱变育种

亚硝基肌(NTG）是一种高效的诱变剂,对 细菌^[1,3,4]、酵母菌^[5]、、黑曲霉^[2]、、天蓝色链霉菌^[6]、 等的诱变效果显著。为了提高卡那霉素生产菌 种的发酵单位,我们研究了在pH6的条件下,不 同缓冲液、NTG不同浓度和不同处理时间对卡 那霉素链霉菌的杀菌率和效价变异频率的影 响,找到了最适诱变条件,先后筛选到两株高产 突变株K-102和K-41,并已用于生产。     

Induction of cytoplasmic petite in yeast by guanidine hydrochloride: Combined treatment with other inducing agents

We have studied the induction of ?^? mutants by guanidine hydrochloride (GuHCl) in combination with other known inducers: ethidium bromide (EB), berenil and ultraviolet light. Competition was observed when cells were simultaneously treated with optimal concentrations of EB and GuHCl; on the other hand, treatment of cells with EB in the presence of non-inducing concentrations of GuHCl resulted in the stimulation of ?^? induction by EB. Furthermore, using a strain which upon treatment with high EB concentrations shows recovery of respiratory competence, the presence of GuHCl did not interfere either with the early phase of induction or with the recovery phase, but it did interfere in a competitive fashion with the final irreversible phase of EB induction. In the case of berenil, a synergistic effect was seen when cells were pretreated with GuHCl. A synergistic induction was also observed when cells were submitted to UV prior to GuHCl treatment. These results suggest that GuHCl, EB and berenil act via some common step in their ?^? induction pathways. Moreover, GuHCl may somehow be decreasing the efficiency of dark repair of ultraviolet lesions on mitochondrial DNA.     

搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化

目的：摸索搅拌式生物反应器培养小鼠胚胎干细胞（mESC）的最佳条件,建立一种批量制备拟胚体（EB）的方法。方法：研究mESC不同接种密度及生物反应器初始搅拌速度对EB形成的数量和质量的影响,以细菌培养皿中形成的EB为对照,用抗坏血酸诱导其向心肌细胞分化,比较两种培养体系对EB心肌细胞分化潜能的影响,通过免疫荧光染色及RT PCR对ESC来源的心肌细胞进行鉴定。结果：当mESC接种密度为1×105~3×105个/ml,搅拌速度设定为15~30r/min时,搅拌式生物反应器能高效制备出大量相对均一的EB,EB中几乎没有坏死细胞。与细菌培养皿制备的EB相比,生物反应器培养的EB向心肌细胞分化的效率更高,并表达心肌特异性基因。结论：搅拌式生物反应器培养促进EB的形成及其向心肌细胞分化,是一种更为理想的EB培养系统。     

Induction of sexual behavior in ovariectomized rhesus females with 19-hydroxytestosterone

Eight sexually experienced long-term ovariectomized female rhesus monkeys were given tests of sexual behavior following treatment with 19-hydroxytestosterone (19-OH-T, 1 mg/day for 13 days), and their performance was compared with that following treatment with estradiol benzoate (EB, 10 μg/day for 13 days). Each female was tested for 10 min with each of nine adult males. Blood samples were taken on the last day of treatment with EB, at the end of the intervening 3-month period of no treatment, and on the last day of treatment with 19-OH-T. Blood levels of testosterone and estradiol were quantified by radioimmunoassay. Mean rate of presenting at a distance (proceptive behavior) was significantly higher (P < 0.05) when they were treated with 19-OH-T, but the ratio of presents to male contacts (receptive behavior) was significantly higher (P <0.05) when they were treated with EB. All other components of female sexual behavior were the same. Males displayed fewer annoyance responses (rejecting jerk, P < 0.05) when the females were treated with 19-OH-T than when they were treated with EB. All other male responses occurred with the same frequencies under the two female treatment conditions. Injection of 19-OH-T and EB both resulted in plasma testosterone and estradiol levels higher than those found in the untreated condition. Testosterone levels did not differ under the two treatments (P > 0.05), but estradiol levels were higher under EB treatment than under 19-OH-T (P < 0.05). This study suggests that both testosterone and estradiol are essential for maximum sexual performance and that various components of sexual behavior may be differentially influenced by the ratio of testosterone to estradiol in plasma.     

Role of erythropoietin for angiogenesis and vasculogenesis: from embryonic development through adulthood

Erythropoietin (EPO), a stimulator of erythropoiesis, was previously shown to stimulate angiogenesis and proliferation of endothelial cells. Here, we investigated and compared the influence of EPO on cell number, proliferation, apoptosis, migration, and differentiation of endothelial cells in intact mouse embryoid bodies (EB), isolated endothelial cells from EB (EBEC), and adult human endothelial progenitor cells (hEPC). EB were treated with EPO (0.5 U/ml) immediately after plating was completed (day 5+0) or 3 days later. EPO treatment was continued until days 5+3 or 5+6. Cultured EBEC were treated 3 days after being plated, and primary hEPC from young healthy adults were treated 5 days after being plated with EPO for 48 h. Immunohistochemistry was performed with anti-PECAM (CD31), anti-Ki67, anti-CD34, anti-CD133, anti-EphB4, and anti-ephrinB2 antibodies. In all, mouse EB and EBEC and hEPC, EPO-treatment resulted in increased number of endothelial cells, increased proliferation, decreased apoptosis, and enhanced migration. In EB, this EPO effect was most pronounced when treatment was begun early (day 5+0) and was accompanied by an enhanced endothelial tube formation. In EBEC and hEPC, EPO shifted the phenotypic differentiation toward an increased ratio of EphB4-positive cells, i.e., toward a venous phenotype. These results are consistent with an important role of EPO for the number, proliferation, apoptosis, function, and phenotypical development of immature endothelial cells, which persists from early development through adulthood. They provide additional and further evidence for a strong interrelation between hematopoiesis and vasculogenesis/angiogenesis (sharing the same pathways), which may be important in many physiological and pathophysiological conditions.     

应用旋转生物反应器批量制备拟胚体的研究

以小鼠胚胎干细胞(ES-D3)为模型,应用新型细胞培养系统——STLV型旋转生物反应器(rotarycellculturesystem,RCCS)建立一种批量制备拟胚体(embryoidbodies,EBs)的新方法,研究不同细胞接种密度及培养时间对RCCS内EBs产生效率的影响。为了进一步研究该制备方法是否对EBs的分化潜能产生影响,对照传统方法制备的EBs,利用形态学及RT-PCR方法测定经旋转生物反应器制备的EBs在自发性或诱导条件下(1%DMSO)向心肌细胞的分化能力。结果表明:ES-D3在RCCS内能够高效形成EBs,与传统的直接悬浮法比较,其EBs的形成效率可达到后者的2倍。1×104个/ml为最佳细胞接种密度,培养时间也是在RCCS制备EBs过程中的重要因素之一,培养第4~5天为最佳收获EBs的时间。与悬滴法制备的EBs比较,该方法制备的EBs分化为心肌细胞的潜能未改变。由此,应用旋转生物反应器可以高效制备EBs,该方法制备的EBs可以用于发育生物学等基础及应用领域的相关研究。     

P19 Embryonal carcinoma cells: a new model for the study of endothelial cell differentiation

P19 embryonal carcinoma cells were examined under different conditions of embryoid body (EB) formation to evaluate endothelial differentiation. A prominent capillary-like network including PECAM-1 positive cells and upregulated expression of endothelial markers Tie-1, Tie-2, Flk-1 and PECAM-1 were identified in the DMSO-treated group during EB formation as well as after VEGF treatment. About 4% of the P19 cells differentiated into PECAM-1 positive cells on day 14 of differentiation. These results suggest that the P19 cells have the potential for endothelial cell differentiation and provide a new model system for future research.     

Synergistic effect of retinoic acid and vitamin D analog EB1089-induced apoptosis of hepatocellular cancer cells

Previous report showed that leukemia cells’ differentiation could be induced by retinoic acid (RA), and prostate cancer cells’ proliferation could be inhibited by Vitamin D or its analog. This study aimed to examine whether RA and vitamin D analog EB1089 have synergistic effect on hepatocellular cancer cells’ apoptosis. The hepatocellular cancer cell lines’ viability was determined by MTT method after treating by RA and EB1089 alone or in combination, cell cycle of SSMC-7721 cell analyzed by FACS, mitochondrial membrane potential of SSMC-7721 under different treatments were detected using MitoTracker Red CMXRos. TUNEL analysis was also used for cell apoptosis detection. Real time-PCR and Western Blot assay were used to detect the expression of Bcl-2 and Bax. Moreover, hepatocellular cancer model was developed by subcutaneously (S.C.) challenging H22 cells to nude mice. In the combination group (10 μmol/L RA, 10 nmol/L EB1089), the viability of hepatocellular cancer cells decreased significantly compared with drugs used alone (P < 0.05). From the TUNEL analysis, SSMC-7721 cells have a higher apoptotic ratio in the combined drug group than in the groups for which the drugs were used separately. In a hepatocellular cancer model, the tumor weight of H22 tumor bearing mice was more reduced in the combined drug treated group when compared to the groups for which the drugs were used alone (P < 0.05), in addition, significantly prolonged survival was observed. Combination of RA and EB1089 exert synergistic growth inhibition and apoptosis induction on hepatocellular cancers cells.     

Electron Microscope Observations on the Effects of Polymixin B Sulfate on Cell Walls of Chlamydia psittaci

The effects of polymixin B sulfate on cell walls of mature elementary body (EB) and of immature developmental reticulate body (RB) of Chlamydia psittaci were investigated. When purified EB were treated with polymixin (10(4) units per ml or more) at 37 C for 60 min, about 70% of EB was found to be covered with a number of projections. Further incubation did not increase the percentage affected. The infectivity after treatment as assayed by the inclusion counting technique was reduced by 70% of the original titer. These results suggest that EB with the projections are no longer infective. The projections had obscure outlines and were 20 to 40 nm in diameter when seen in thin sections. In the negatively stained preparations, the projections were composed of aggregations of fine particles 4 to 5 nm in diameter. Treatment with sodium dodecyl sulfate at the same concentration used for cell wall isolation removed the projections completely, and the cell walls were converted to rather ragged forms apparently composed of outside and inside layers. When RB cell walls prepared from infected cells at 18 hr after infection were treated with polymixin at the same concentration, the projections having the same morphology with those seen on treated EB cell walls were observed only on the inside surface of cell wall.     

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning,lateral vessel (STLV) bioreactor

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 × 10⁵ cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.     

An alteration in the hypothalamic action of estradiol due to lack of progesterone exposure can cause follicular cysts in cattle

Many mammals, including cattle, can develop ovarian follicular cysts, but the physiological mechanisms leading to this condition remain undefined. We hypothesized that follicular cysts can develop because estradiol will induce a GnRH/LH surge on one occasion but progesterone exposure is required before another GnRH/LH surge can be induced by estradiol. In experiment 1, 14 cows were synchronized with an intravaginal progesterone insert (IPI) for 7 days, and prostaglandin F(2alpha) was given on the day of IPI removal. Estradiol benzoate (EB; 5 mg i.m.) was given 3 days before IPI removal to induce atresia of follicles. Cows were given a second EB treatment 1 day after IPI removal to induce a GnRH/LH surge in the absence of an ovulatory follicle. All cows had an LH surge following the second EB treatment, and 10 of 14 cows developed a large-follicle anovulatory condition (LFAC) that resembled follicular cysts. These LFAC cows were given a third EB treatment 15 days later, and none of the cows had an LH surge or ovulation. Cows were then either not treated (control, n = 5) or treated for 7 days with an IPI (n = 5) starting 7 days after the third EB injection. Cows were treated for a fourth time with 5 mg of EB 12 h after IPI removal. All IPI-treated, but no control, cows had an LH surge and ovulated in response to the estradiol challenge. In experiment 2, cows were induced to LFAC as in experiment 1 and were then randomly assigned to one of four treatments 1) IPI + EB, 2) IPI + GnRH (100 microg), 3) control + EB, and 4) control + GnRH. Control and IPI-treated cows had a similar LH surge and ovulation when treated with GnRH. In contrast, only IPI-treated cows had an LH surge following EB treatment. Thus, an initial GnRH/LH surge can be induced with high estradiol, but estradiol induction of a subsequent GnRH/LH surge requires exposure to progesterone. This effect is mediated by the hypothalamus, as evidenced by similar LH release in response to exogenous GnRH. This may represent the physiological condition that underlies ovarian follicular cysts.