鞘氨醇-1-磷酸对人脐带间充质干细胞增殖及表面标记物表达的影响

鞘氨醇－1－磷酸（sphingosine－1 phosphate, S1P）是一种脂质信号分子,与细胞增殖、凋亡和迁移等有密切关系．本研究发现,S1P促进人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC－MSCs)增殖,但目前关于其作用信号通路及S1P对hUC－MSCs表面标记表达的影响尚不十分清楚．Real－time PCR检测hUC－MSCs中S1P受体mRNA表达情况,发现在hUC－MSCs中优势表达S1PR1 3,而S1PR4、S1PR5的表达很少．MTT法检测S1PR1/3拮抗剂VPC23019、S1PR2拮抗剂JTE013、S1PR3拮抗剂CAY10444、Gi蛋白抑制剂PTX和ERK抑制剂PD98059对S1P诱导hUC－MSCs增殖的影响．结果显示,VPC23019完全抑制S1P诱导的hUC MSCs增殖,JTE013对此没有明显影响,CAY10444部分抑制S1P诱导的hUC MSCs增殖,PTX、PD98059完全抑制S1P诱导hUC－MSCs增殖．进一步用Western印迹检测ERK1/2磷酸化水平揭示,S1P通过促进ERK1/2磷酸化进而促进hUC MSCs增殖．流式细胞术检测发现,S1P对hUC MSCs表面标记物(CD45、CD34、CD90、CD29、CD105、CD44、CD73、CD71)表达没有明显影响．本研究证明,S1P通过S1PR1/3、Gi偶联蛋白及ERK1/2信号通路促进hUC MSCs增殖,而对hUC MSCs表面标记物表达无明显影响．     

Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity

Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA^NUCB or addAB^NUC mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA^NUCB^NUC double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA^HELB lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA^NUC and addA^HEL mutants colonized the stomach less efficiently than the wild type; addB^NUC showed partial attenuation. E. coli ΔrecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.Infection of the stomach with Helicobacter pylori causes a variety of diseases including gastritis, peptic ulcers, and gastric cancer (1). A central feature of the pathology of these conditions is the establishment of a chronic inflammatory response that acts both on the host and the infecting bacteria (2). Both epithelial (3, 4) and lymphoid (5, 6) cells in the gastric mucosa of infected individuals release DNA-damaging agents that can introduce double-stranded (ds)² breaks into the bacterial chromosome (7). The ds breaks must be repaired for the bacteria to survive and establish chronic colonization of the stomach. Homologous recombination is required for the faithful repair of DNA damage and bacterial survival. Alteration of the expression of one of a series of cell surface proteins on H. pylori occurs by an apparent gene conversion of babA, the frequency of which is reduced in repair-deficient strains (8, 9). This change in the cell surface, which may allow H. pylori to evade the host immune response, is a second means by which recombination can promote efficient colonization of the stomach by H. pylori.The initiation or presynaptic steps of recombination at dsDNA breaks in most bacteria involves the coordinated action of nuclease and helicase activities provided by one of two multisubunit enzymes, the AddAB and RecBCD enzymes (10). Escherichia coli recBCD null mutants have reduced cell viability, are hypersensitive to DNA-damaging agents, and are homologous recombination-deficient (11–14). Similarly, H. pylori addA and addB null mutants are hypersensitive to DNA-damaging agents, have reduced frequencies of babA gene conversion, and colonize the stomach of mice less efficiently than wild-type strains (8).The activities of RecBCD enzyme from E. coli (15–19) and AddAB from H. pylori (8) or Bacillus subtilis (20–23) indicate some common general features of the presynaptic steps of DNA repair. In the case of E. coli, repair begins when the RecBCD enzyme binds to a dsDNA end and unwinds the DNA using its ATP-dependent helicase activities (17, 24). Single-stranded (ss) DNA produced during unwinding, with or without accompanying nuclease, is coated with RecA protein (16, 25). This recombinogenic substrate engages in strand exchange with a homologous intact duplex to form a joint molecule. Joint molecules are thought to be converted into intact, recombinant DNA either by replication or by cutting and ligation of exchanged strands (26).Although the AddAB and RecBCD enzymes appear to play similar roles in promoting recombination and DNA repair, they differ in several ways. RecBCD is a heterotrimer, composed of one copy of the RecB, RecC, and RecD gene products (27), whereas AddAB has two subunits, encoded by the addA and addB genes (21, 28). The enzyme subunit(s) responsible for helicase activity can be inferred from the presence of conserved protein domains or the activity of purified proteins. AddA, RecB, and RecD are superfamily I helicases with six highly conserved helicase motifs, including the conserved Walker A box found in many enzymes that bind ATP (29–32). A Walker A box is defined by the consensus sequence (G/A)XXGXGKT (X is any amino acid (29). RecBCD enzymes in which the conserved Lys in this motif is changed to Gln have a reduced affinity for ATP binding (33, 34) and altered helicase activity (17, 35–37).A nuclease domain with the conserved amino acid sequence LDYK is found in RecB, AddA, AddB, and many other nucleases (38). The conserved Asp plays a role in Mg²⁺ binding at the active site; Mg²⁺ is required for nuclease activity (39). The recB1080 mutation, which changes codon 1080 from the conserved Asp in this motif to Ala, eliminates nuclease activity (39).We have recently shown that addA and addB deletion mutants are hypersensitive to DNA-damaging agents and impaired in colonization of the mouse stomach compared with wild-type strains (8). To determine the roles of the individual helicase and nuclease activities of H. pylori AddAB in DNA repair and infectivity, we used site-directed mutagenesis to inactivate the conserved nuclease domains of addA and addB and the conserved ATPase (helicase) domain of AddA. Here, we report that loss of the AddAB helicase is sufficient to impair H. pylori DNA repair and infectivity and, when the genes are expressed in E. coli, homologous recombination. AddAB retains partial activity in biochemical and genetic assays when either of the two nuclease domains is inactivated but loses all detectable nuclease activity when both domains are inactivated. Remarkably, H. pylori AddAB can produce recombinants in E. coli only in the presence of H. pylori RecA, suggesting a species-specific interaction in which AddAB facilitates the production of ssDNA-coated with RecA protein. Our results show that both the helicase and nuclease activities are required for the biological roles of H. pylori AddAB.     

流式细胞术研究细胞凋亡的方法与技术

细胞发生凋亡时,会伴随着一系列形态学、生物化学及分子生物学性质的变化,包括细胞皱缩,核染色质凝聚,细胞膜通透性改变,Caspases激活,线粒体跨膜电位降低,膜磷酯酰丝氨酸外化,胞质Ca2+浓度升高,DNA片段化及含量变化等特点.应用流式细胞术进行细胞凋亡的研究,对于探讨胚胎发育、衰老以及研究肿瘤的发生、发展和转化等病理生理过程和病毒感染及免疫等具有十分重要的意义.本文就细胞凋亡的特征、基于细胞膜功能的流式细胞术检测方法和基于细胞器功能的流式细胞术检测方法等关键性问题进行了阐述.     

圆瓣姜花根茎挥发油的化学成分

利用水蒸汽蒸馏法提取圆瓣姜花根茎挥发油,运用毛细管气相色谱-质谱联用法对挥发油进行了分析,分离出60个峰,鉴定了其中的51种成分,所鉴定成分占挥发油总量的97.32%,其主要化学成分为单萜及倍半萜类化合物。     

大鼠子宫内膜异位模型的建立与组织学观察

目的为开发诊治子宫内膜异位症(endometriosis,EMT)的新药研究提供理想的动物模型。方法取雌性未交配性成熟大鼠,术前雌激素诱导,麻醉开腹取部份右侧子宫,将内膜种植于左腹壁内,术后16周取出包块,进行组织形态学、组织化学观察。结果异位内膜在腹壁内生长,呈隆起囊状小包块,内有黏液,具有正常子宫内膜基本组织结构,囊腔较大。异位内膜中有糖原、RNA的存在。结论该手术方法建立的子宫异位内膜生长良好,术后一周就可摸及包块大小,为开发研究子宫内膜异位症的新药提供了方便。     

Antimicrobial activity and phytochemical analyses of Polygonum aviculare L. (Polygonaceae), naturally growing in Egypt

Polygonum aviculare (Polygonaceae) is an herb commonly distributed in Mediterranean coastal regions in Egypt and used in folkloric medicine. Organic and aqueous solvent extracts and fractions of P. aviculare were investigated for antimicrobial activities on several microorganisms including bacteria and fungi. Phytochemical constituents of air-dried powered plant parts were extracted using aqueous and organic solvents (acetone, ethanol, chloroform and water). Antimicrobial activity of the concentrated extracts was evaluated by determination of the diameter of inhibition zone against both Gram-negative and Gram-positive bacteria and fungi using paper disc diffusion method.Results of the phytochemical studies revealed the presence of tannins, saponins, flavonoids, alkaloids and sesquiterpenes and the extracts were active against both Gram-negative and Gram-positive bacteria. Chloroform extract gave very good and excellent antimicrobial activity against all tested bacteria and good activity against all tested fungi except Candida albicans. Structural spectroscopic analysis that was carried out on the active substances in the chloroform extract led to the identification of panicudine (6-hydroxy-11-deoxy-13 dehydrohetisane).Evaluation of the antimicrobial activity of panicudine indicated significant activity against all tested Gram-negative and Gram-positive organisms. Panicudine displayed considerable activity against the tested fungi with the exception of C. albicans. Antimicrobial activity of the extracts was unaffected after exposure to different heat treatments, but was reduced at alkaline pH. Studies of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of panicudine on the tested organisms showed that the lowest MIC and the MBC were demonstrated against Salmonella paratyphi, Bacillus subtilis and Salmonella typhi and the highest MIC and MBC were against Staphylococcus aureus.     

Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.     

In vivo gene transfer of Kv1.5 normalizes action potential duration and shortens QT interval in mice with long QT phenotype

Mutations in cardiac voltage-gated K+ channels cause long QT syndrome (LQTS) and sudden death. We created a transgenic mouse with a long QT phenotype (Kv1DN) by overexpression of a truncated K+ channel in the heart and investigated whether the dominant negative effect of the transgene would be overcome by the direct injection of adenoviral vectors expressing wild-type Kv1.5 (AV-Kv1.5) into the myocardium. End points at 3-10 days included electrophysiology in isolated cardiomyocytes, surface ECG, programmed stimulation of the right ventricle, and in vivo optical mapping of action potentials and repolarization gradients in Langendorff-perfused hearts. Overexpression of Kv1.5 reconstituted a 4-aminopyridine-sensitive outward K+ current, shortened the action potential duration, eliminated early afterdepolarizations, shortened the QT interval, decreased dispersion of repolarization, and increased the heart rate. Each of these changes is consistent with a physiologically significant primary effect of adenoviral expression of Kv1.5 on ventricular repolarization of Kv1DN mice.