A method for isolation of a large amount of a single-stranded DNA fragment

Single-stranded DNA inserts can be digested from recombinant phage DNA of M13mp7 with BamHI or EcoRI restriction endonucleases. The single-stranded DNA is satisfactory for DNA sequencing and nuclei acid hybridization.     

Effects of synthetic and naturally occurring flavonoids on metabolic activation of benzo[a]pyrene in hamster embryo cell cultures.

Biochanin A, an isoflavone, has previously been shown to inhibit the metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) to metabolites that bind to DNA in hamster embryo cells and are mutagenic in Chinese hamster V79 cells. To determine the structural features required for this activity and to attempt to find more effective inhibitors, a series of synthetic and naturally occurring flavonids were tested for their ability to modulate B[a]P metabolism in hamster embryo cell cultures. The observed structure-activity relationships indicate that the structural features of flavonoids important for effective inhibition of B[a]P metabolism in hamster embryo cells are the presence of two hydroxyl, two methoxyl, or methyl and hydroxyl substituents at the 5- and 7-positions and a 2,3-double bond. Flavones are slightly better inhibitors of B[a]P metabolism than the corresponding isoflavones. A substituent at the 4'-position is not essential for inhibition of B bdP metabolism. The presence of a hydroxyl group at position 3 slightly enhances activity. Apigenin, acacetin and kaempferide are effective inhibitors of B[a]P-induced mutagenesis in a hamster embryo cell-mediated V79 cell mutation assay. However, apigenin is cytotoxic at the inhibitory dose, whereas acacetin and kaempferide are not. These results suggest that acacetin and kaempferide are promising candidates for in vivo testing as potential chemopreventive agents.     

Intracellular protein phosphorylation in oat (Avena sativa L.) protoplasts by phytochrome action: involvement of protein kinase C

Phosphorylations of two proteins (27 KDa, 32 KDa) in oat cells were dependent on phytochrome action. To determine which kinase system(s) for the phosphorylation of these two proteins are controlled by the phytochrome, involvement of the Ca2+/DG dependent protein kinase (protein kinase C) was first investigated. When a protein kinase C inhibitor (1-(5-isoquinoline sulfonyl)-2-methylpiperazine:H-7) or the inositol phospholipid metabolic blocker Li+ was added into the cell suspension, respectively, the phosphorylations of these two proteins were substantially reduced. On the other hand, an addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG:activator of protein kinase C) or phorbol 12-myristate 13-acetate (TPA: tumor promoting phorbol ester) enhanced the phosphorylations of these proteins. These results suggest that phytochrome action is certainly connected with the protein phosphorylation via the activation of protein kinase C or a similar molecule with protein kinase C.     

Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI, active in mutation-specific reversion, and uvsC, a recA homolog, required for all UV mutagenesis

Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function. To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation. Spontaneous and induced frequencies were compared in forward and reverse mutation assays. All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis. The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations. In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis. Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate). Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus. In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli.     

长裙竹荪抑菌活性物质的分离纯化及其抑菌效果

长裙竹荪Dictyophora indusiata是珍贵的食药用真菌,具有很强的抑菌作用,在天然防腐剂开发方面具有广阔的应用前景。本研究以长裙竹荪的抑菌活性为指标,通过萃取、3次不同流动相的硅胶柱层析、1次反相柱层析和薄层层析法对竹荪提取物进行分离纯化,得到一个抗菌活性强的单体化合物。根据核磁共振波谱等数据分析,推断该化合物为间苯三酚。以巨大芽孢杆菌和肠炎沙门氏菌为供试菌,用平板打孔法及原位抑菌法测定该化合物的抑菌效果,结果表明：该化合物对这两种菌有很强的抑制作用,半抑制浓度分别为83.06μg/mL和51.58μg/mL。本研究首次从长裙竹荪中获得具有抗菌活性的单体化合物间苯三酚,为竹荪天然抗菌物质的开发提供理论依据。     

An Antiaging Electrolyte Additive for High‐Energy‐Density Lithium‐Ion Batteries

High‐capacity Li‐rich layered oxide cathodes along with Si‐incorporated graphite anodes have high reversible capacity, outperforming the electrode materials used in existing commercial products. Hence, they are potential candidates for the development of high‐energy‐density lithium‐ion batteries (LIBs). However, structural degradation induced by loss of interfacial stability is a roadblock to their practical use. Here, the use of malonic acid‐decorated fullerene (MA‐C₆₀) with superoxide dismutase activity and water scavenging capability as an electrolyte additive to overcome the structural instability of high‐capacity electrodes that hampers the battery quality is reported. Deactivation of PF₅ by water scavenging leads to the long‐term stability of the interfacial structures of electrodes. Moreover, an MA‐C₆₀‐added electrolyte deactivates the reactive oxygen species and constructs an electrochemically robust cathode‐electrolyte interface for Li‐rich cathodes. This work paves the way for new possibilities in the design of electrolyte additives by eliminating undesirable reactive substances and tuning the interfacial structures of high‐capacity electrodes in LIBs.     

脑缺血大鼠肺组织NGF/TrkA表达变化及其意义

目的:探讨大鼠脑缺血后肺组织神经生长因子(nerve growth factor,NGF)/酪氨酸蛋白激酶A(tropomyosin-related kinase A,TrkA)表达的变化。方法:成年雄性大鼠随机分为假手术组和脑缺血后6 h、24 h及48 h组,每组8只。建立大脑中动脉永久性局灶性缺血模型,术后于各时间点麻醉处死大鼠后,测定肺组织湿重/干重(W/D),光镜下观察HE染色肺组织病理学改变;Western blot法检测肺组织NGF、TrkA蛋白表达。结果:与假手术组相比,脑缺血后6 h肺组织W/D有增加,差异无统计学意义(P>0.05),而脑缺血后24 h及48 h肺组织W/D均有显著增高(P<0.05),其中以24 h组最明显(P<0.01);脑缺血后6 h肺组织出现轻度充血、水肿及炎性改变,24 h及48 h组肺泡结构破坏明显。病理学评分结果显示,脑缺血后24 h及48 h组大鼠肺组织病理评分较假手术组显著升高(P<0.05);Western blot法显示,脑缺血6 h肺组织中NGF表达增加(P<0.05),48 h时表达有下降趋势,但仍高于假手术组(P<0.05)。而肺组织中TrkA表达在脑缺血6h有下降(P>0.05),24 h下降明显(P<0.05),48 h时TrkA蛋白表达虽有上升,仍显著低于假手术组(P<0.05)。结论:大鼠肺组织NGF/TrkA的动态变化可能参与了脑缺血后肺损伤的病理生理过程。     

基于磁驱动技术的空肠营养管的设计

目的:为解决目前床旁鼻肠管快速置入成功率低这一临床难题,该文提出一种基于磁驱动技术的空肠营养管的设计。方法:分析了现有空肠营养管置管过程中的动力因素,结合磁驱动技术原理,提出了通过体外旋转磁场带动体内感应磁体螺旋式前进的设计方案,以期缩短空肠营养管的飘管时间。结果:该设计包括磁性空肠营养管和体外磁力驱动装置两部分。其中磁性空肠营养管由管体和感应磁头两部分构成,感应磁头包括磁体内核和硅胶外壳。磁力驱动装置由多极磁体和手持式微型电机组成。操作时通过体外磁力驱动装置发出大旋转磁场带动空肠营养管的感应磁头做轴向旋转运动,可加速空肠营养管在肠道内的移动,达到缩短飘管时间的目的。结论:该设计基于磁驱动技术原理,设计巧妙,符合磁力学原理,操作简单,具有临床应用潜力。     

miR-195通过靶向调控趋化因子5促进胃癌细胞增殖、转移及侵袭的实验研究

目的:研究miR-195通过靶向调控趋化因子5促进胃癌细胞增殖、转移及侵袭的分子机制。方法:选取MGC803及NCI-N87细胞,根据转染不同分为:miR-NC组(空质粒),miR-195-mimics组(模拟序列)。实时荧光定量PCR法检测miR-195表达;MTT检测细胞增殖能力;Transwell侵袭实验检测细胞侵袭力;细胞划痕实验检测细胞转移能力;流式细胞术检测细胞凋亡情况;Western blot检测chemokine 5表达水平;Spearman相关分析miR-195及chemokine 5相关性。荧光素酶实验验证miR-195与chemokine 5的靶向关系。结果:miR-195-mimics组细胞miR-195水平高于miR-NC组(P0.05);miR-195 mimics组第1、2、3、4 d细胞活力低于miR-NC组(P0.05);miR-195 mimics组G1细胞高于miR-NC组,G2期、S期细胞低于miR-NC组,G2/S期细胞比值低于miR-NC组(P0.05);miR-195 mimics组划痕距离大于miR-NC组(P0.05);miR-195 mimics组细胞侵袭数低于miR-NC组(P0.05);miR-195-mimics组细chemokine 5蛋白含量低于miR-NC组(P0.05);miR-195 m RNA水平与chemokine 5蛋白含量负相关(r=-0.398,P=0.00);miR-195可直接靶向chemokine 5。结论:miR-195可通过靶向chemokine 5促进胃癌MGC803及NCI-N87细胞的增殖、转移及侵袭。     

达格列净对高渗诱导的血管内皮细胞衰老的干预研究

目的:探讨SGLT2i类药物达格列净(dapagliflozin)对高渗诱导的人脐静脉内皮细胞(HUVECs)衰老的影响。方法:将HUVECs分为空白组(Blank组)、高渗330组(M-330组)、高渗350组(M-350组)、达格列净+高渗组(DAPA+M-350组),高渗培养环境由甘露醇诱导。衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老情况;免疫荧光染色检测SGLT2表达变化;Western blot检测SGLT2、细胞衰老标志物p21的表达变化,JC-1染色试剂盒检测线粒体膜电位的变化。结果:免疫荧光染色和western blot结果显示,Blank组,M-330组及M-350组细胞上均存在SGLT2受体蛋白表达,且Blank组,M-330组及M-350组的SGLT2表达依次显著增加。与Blank组相比,M-350组SA-β-Gal胞质蓝染、染色阳性率、衰老蛋白p21及SGLT2表达显著增加,并伴有线粒体膜电位的显著下降(P0.05);DAPA+M-350组与M-350组相比,SA-β-Gal胞质蓝染、染色阳性率和p21表达显著下降,并伴有线粒体膜电位的显著上升(P0.05)。结论:HUVECs上存在SGLT2受体蛋白,且在300-350 m Osm/L范围内随着渗透压的升高而增加,达格列净可改善高渗所诱导的血管内皮细胞衰老,其机制可能与达格列净改善高渗导致的线粒体功能障碍有关。