The Effect of Oxygen on Melanin Precursors Released from Retinal Pigment Epithelial Cells In Vitro

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240–275 nm and a maximum around 300 nm wth a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Cleavage of poly(ADP-ribose) polymerase during apoptosis induced by nicotinamide at high concentrations in tobacco suspension cells

Apoptosis induced by high concentrations of nicotinamide in tobacco suspension cells was observed. When cells were treated with 250 mM nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180–200 bp, condensation and peripheral distribution of nuclei chromatin and positive reaction to the TUNEL assay. In addition, the degradation of poly (ADP-ribose) polymerase (PARP) was also detected. This indicates that caspase-3-like activity is involved in apoptosis in cultured tobacco cells induced by high-concentration nicotinamide. However, as an inhibitor of PARP, nicotinamide has a contrary effect on apoptosis at low concentrations, which suggests that nicotinamide plays a dual role depending on to its concentration in cells.     

Agonist Effects on the Intracellular Cyclic AMP Concentration of Retinal Pigment Epithelial Cells in Culture

Accumulation of cyclic AMP in intact cultured pigment epithelial cells was rapidly enhanced by several agonists. These included vasoactive intestinal peptide (100-fold), glucagon (fivefold), thyroid-stimulating hormone (threefold), prostaglandin E1 (24-fold), L-isoproterenol (27-fold), and histamine (fourfold). The rapidity and magnitude of these effects suggest that these agonists may regulate important retinal pigment epithelial cell functions.     

蟾毒灵对人卵巢癌SKOV-3细胞增殖的影响

目的：探讨蟾毒灵对人卵巢癌SKOV-3细胞增殖抑制和凋亡的影响,为卵巢癌临床治疗提供依据和分子基础。方法：不同浓度蟾毒灵处理卵巢癌SKOV-3细胞后,MTT法检测细胞增殖抑制作用,细胞免疫化学染色法检测细胞的凋亡,Western Blot法检测Bax,Bcl-2,Caspase-3蛋白以及计算Bax／Bcl-2的比值。结果：蟾毒灵能够抑制SKOV-3细胞的增殖,且成时间和剂量依赖性,免疫荧光显示蟾毒灵对SKOV-3细胞具有凋亡作用,Western Blot检测发现蟾毒灵能够促进Caspase-3蛋白的活化,提高Bax／Bcl-2的比值。结论：蟾毒灵在体外能够抑制卵巢癌SKOV-3细胞的增殖和促进卵巢癌SKOV-3细胞的凋亡。     

蟾毒灵对人卵巢癌SKOV-3 细胞增殖的影响

目的：探讨蟾毒灵对人卵巢癌SKOV-3细胞增殖抑制和凋亡的影响,为卵巢癌临床治疗提供依据和分子基础。方法：不同浓 度蟾毒灵处理卵巢癌SKOV-3 细胞后,MTT 法检测细胞增殖抑制作用,细胞免疫化学染色法检测细胞的凋亡,Western Blot 法检 测Bax,Bcl-2,Caspase-3 蛋白以及计算Bax/Bcl-2 的比值。结果：蟾毒灵能够抑制SKOV-3 细胞的增殖,且成时间和剂量依赖性, 免疫荧光显示蟾毒灵对SKOV-3 细胞具有凋亡作用,Western Blot 检测发现蟾毒灵能够促进Caspase-3 蛋白的活化,提高 Bax/Bcl-2 的比值。结论：蟾毒灵在体外能够抑制卵巢癌SKOV-3 细胞的增殖和促进卵巢癌SKOV-3 细胞的凋亡。     

不同培养条件对鸡胚胎生殖细胞Oct4启动子DNA甲基化的影响

旨在在体外通过胚胎生殖细胞(EGC)和细胞外基质共同构建一个EGC的小生境(niche)模型,通过比较Oct4DNA甲基化的变化,研究niche中特有的表观遗传效应对胚胎生殖细胞自我更新的影响.构建EGC细胞标准培养方案(对照组)和改良培养方案(试验组),采用甲基化特异性PCR( MS-PCR)技术、RT-PCR技术,对比分析两种培养方案中Oct4启动子的甲基化状态,判断基因表达与其CpG岛甲基化的关系,分析DNA甲基化模式与EGC细胞自我更新的关系.结果显示,试验组可扩增出Oct4的非甲基化扩增产物,而对照组为其甲基化扩增产物,试验组Oct4表达水平明显高于对照组.试验组EGC生长状态明显好于对照组.试验表明,在改良培养条件下,Oct4基因启动子DNA甲基化程度较低,且与基因表达水平呈负相关,更有利于维持胚胎生殖细胞的自我更新状态.     

白藜芦醇对紫外线照射后人角质形成细胞AQP3 表达的影响

目的:探讨白藜芦醇对紫外线照射后人皮肤角质形成细胞水通道蛋白3(AQP3)表达的影响及意义。方法:原代培养人皮肤角质形成细胞,采用UVB(20mJ/cm2,40mJ/cm2)照射角质形成细胞后,立即加入0.1mmol/L的白藜芦醇进行干预。RT-PCR检测照射前后角质形成细胞中AQP3 mRNA的表达量,并用羟胺法、比色法、TBA法检测照射前后细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量。结果:1.UVB照射后角质形成细胞AQP3 mRNA的表达量下降(P<0.05),且UVB照射剂量越大,AQP3 mRNA下降越显著(P<0.05)。2.白藜芦醇能显著增加UVB照射后角质形成细胞SOD和GSH-Px活性,并降低细胞MDA含量(P<0.05)。3.白藜芦醇能显著抑制UVB导致的角质形成细胞AQP3 mRNA下降(P<0.05)。结论:白藜芦醇可能通过抑制UVB导致的AQP3 mRNA下降,及提高氧化酶活性、清除自由基的功能,从而延缓皮肤衰老。     

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.     

The Function of SeMNPV IAP3 in Mammalian Cells

The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I/Bam H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in HEK293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in HEK293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.     

Effect of poly(3-hydroxybutyrate) (PHB) content on the starvation-survival of bacteria in natural waters

Abstract The effect of poly(3-hydroxybutyrate) (PHB) content on the survival of wild-type strains and PHB negative mutants of Bacillus megaterium and Alcaligenes eutrophus in natural waters was studied. The survival strategy of B. megaterium was dominated by the development of resistant forms, but the number of the wild-type vegetative cells was higher than that of PHB mutant strain. In some environmental conditions the mutant spores needed a heat shock for germination, a fact that suggests, for the first time, that PHB plays a role in this phenomenon. Survival of A. eutrophus wild-type strain in all experiments was higher compared to the PHB mutant, and differences were significant. In raw river water, survival of both species was lower than in sterile river water.     

beta-榄香烯对卵巢癌细胞SKOV3 增殖和凋亡的影响

目的:探讨β-榄香烯对卵巢癌细胞SKOV3增殖和凋亡的影响。方法:体外培养卵巢癌SKOV3细胞,将β-榄香烯(浓度梯度为25,50,100,150,200μg/m L),单独作用于卵巢癌SKOV3细胞,加药24 h、48 h后用噻唑蓝(MTT法)法检测细胞增殖情况;用流式细胞术检测加药24 h后对细胞凋亡的影响。结果:1MTT法结果显示β-榄香烯单独用药24 h、48 h后,与对照组相比,实验组对卵巢癌SKOV3细胞的抑制率均高于对照组(P<0.05),并且在一定程度上呈浓度和时间依赖性。2流式细胞术检测显示,β-榄香烯能够促进SKOV3细胞的凋亡。结论:β-榄香烯能够抑制卵巢癌细胞SKOV3的增殖,促进其凋亡。     

3-Aminobenzamide,a potent inhibitor of poly (ADP-ribose) polymerase,causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.     

opn1lw2基因在红光诱导斑马鱼皮肤色素细胞形成中的作用

为了探究感红光视蛋白2基因opn1lw2在红光诱导斑马鱼(Danio rerio)皮肤色素细胞形成中的作用,针对AB品系野生型斑马鱼利用CRISPR/Cas9基因编辑技术敲除感红光视蛋白2基因opn1lw2,构建opn1lw2缺失的纯合opn1lw2-/-品系.使用光强(800±100)lx的红光LED灯(每天光照24...     

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.     

酪氨酸磷酸酶PRL-3增加胃癌细胞BGC823的SP细胞比例并提高其对化疗药物的耐受性

蛋白酪氨酸磷酸酶PRL-3是近年发现的蛋白酪氨酸磷酸酶家族成员,能促进肿瘤细胞的侵袭、转移及上皮细胞间质转型,提示PRL-3可能在肿瘤发生发展及诱导肿瘤干细胞生成中发挥重要作用.由于侧群(SP)细胞具有许多干细胞的性质,SP细胞分选是目前筛选和分离获得干细胞或前体细胞常用的有效方法.为探讨PRL-3在诱导干细胞生成中的潜在作用,本文在建立过表达PRL-3的人胃癌细胞BGC823的基础上,通过SP分选和CCK-8的方法分析PRL-3对BGC823细胞中SP细胞的比例以及对化疗药物耐受性的影响.结果提示,高表达PRL-3提高BGC823中SP细胞的比例（2.5% vs 9.4%）,同时增加BGC823对化疗药物紫杉醇和顺铂的耐受性（相对于对照细胞,其耐药指数分别为1.75和1.29）.由于SP细胞的产生和细胞耐药性的提高与ABC家族基因表达水平上调密切相关,通过定量 RT-PCR和Western印迹检测发现,PRL-3能上调ABCB1和ABCG2的表达.上述研究结果表明,PRL-3有可能通过上调ABCB1和ABCG2的表达,增加胃癌细胞BGC823的SP细胞比例并增加其对化疗药物的耐受性.     

细胞因子对GH3细胞中人生长激素基因表达的影响

为了研究细胞因子IL 11、睫状神经营养因子 (CNTF)和转化生长因子 (TGF β)对大鼠垂体GH3 细胞中人生长激素 (hGH)的基因启动子活性的影响及其与垂体特异性转录因子Pit 1蛋白的关系 ,首先建立含hGH基因启动子 (- 4 84～ 30bp)和荧光素酶融合基因的稳定转化GH3 细胞系 ,然后用细胞因子刺激 ,检测细胞培养液和细胞裂解液中GH的含量 ,反映它们对GH分泌和合成的影响 ;检测GH3 细胞内荧光素酶的变化 ,说明细胞因子对hGH基因启动子活性的作用。将Pit 1蛋白表达质粒 (pcDNA pit 1 cDNA)单独转染或与Pit 1反义寡核苷酸 (Pit 1OND)共转染于稳定转化的GH3 细胞中 ,观察加入细胞因子后荧光素酶的变化 ,探讨细胞因子的作用与Pit 1蛋白的关系。结果表明 ,IL 11(2 0nmol/L)、CNTF(10nmol/L)能刺激大鼠垂体GH3 细胞中GH的分泌和合成 ,增强GH3 细胞中荧光素酶的表达 ,分别增加到对照组的 12 6 %、136 %。TGF β(5nmol/L)能减少GH的分泌和合成 ,抑制荧光素酶的表达到对照组的 77%。Pit 1蛋白过表达和表达被抑制对细胞因子的调节作用没有影响。这说明IL 11、CNTF和TGF β可通过调节大鼠垂体GH3 细胞中hGH基因启动子活性影响GH的合成 ,Pit 1蛋白可能不参与这些调节作用。     

Effect of alcohol feeding mode on the biosynthesis of poly (3-hydroxybutyrate-co-3-hyroxyvalerate)

An alcohol utilizing Alcaligenes eutrophus produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer under phosphate limitation. Fermentation was performed for 42-46 h in a computer-controlled 5-L working volume fed-batch fermentor using ethanol and propanol as carbon sources. The culture experienced phosphate limitation in approximately 19 h. When propanol was used as a sole carbon source, 24 g/L of copolymer with 36.5 mol % of hydroxyvalerate (HV) was produced at a polymer yield of 0.41 g polymer/g alcohol (g/g) and an average polymer production rate of 0.08 g polymer/g residual biomass-h (g/g-h). Two experiments switching alcohol after phosphate exhaustion resulted in better polymer production (g/L), polymer yield (g/g) on alcohol, HV yield (g/g) on propanol, and average polymer production rate (g/g-h) as compared to propanol run without alcohol switching. One switching experiment was from a mixture of 50% ethanol and 50% propanol to 100% propanol and the other experiment was from 100% ethanol to a mixture of 65% ethanol and 35% propanol. Polymer yield for these two experiments was 0.51 g/g and 0.46 g/g, respectively. However, HV mol % in the copolymer for these two runs (30.8 mol % 12.6 mol % respectively) was lower compared to propanol run without alcohol switching (3605 mol %). Direct switch from ethanol to propanol did not support cell growth and polymer production. Polymer production rate and polymer yield changed with time, and the pattern was dependent upon the alcohol feeding mode. (c) 1994 John Wiley & Sons, Inc.     

小豆蔻明调控自噬抑制卵巢癌SKOV3 细胞增殖的研究

目的：卵巢癌为女性常见病,死亡率高,分子靶向治疗药物较少,研发高效低毒的靶向新药意义重大。细胞自噬（autophagy） 维持着细胞内稳态和生长,是抗癌药物作用的关键靶部位。本课题旨在明确小豆蔻明（Cardamonin,CAR）调控自噬对卵巢癌 SKOV3 细胞增殖的影响。方法：体外培养卵巢癌SKOV3 细胞,不同药物分组处理,荧光显微镜下观察单黄酰戊二胺（MDC）染色 后细胞自噬囊泡,Western Blot 法检测细胞自噬相关蛋白LC3的表达,四氮唑蓝（MTT）法观察SKOV3 细胞增殖情况,流式细胞 术检测SKOV3 细胞凋亡的变化。结果：自噬抑制剂3-MA 显著性降低SKOV3 细胞内MDC染色的荧光颗粒数目、LC3Ⅱ蛋白表 达,抑制细胞增殖;而CAR（高、中剂量）、雷帕霉素和AZD8055 与3-MA 联用后细胞内MDC荧光颗粒数目增多、LC3Ⅱ蛋白表达 增加、细胞增殖抑制率及凋亡率明显升高;且高剂量CAR（10-5mol·L-1）的作用比低剂量CAR（10-6mol·L-1）明显。结论：CAR能够 抑制SKOV3 细胞增殖,诱导细胞自噬,促进细胞凋亡。CAR有望成为卵巢癌药物治疗的先导化合物。本研究为进一步研发此类 化合物提供了实验依据及一定的理论基础。