内在化的耐甲氧西林金黄色葡萄球菌诱导宿主细胞凋亡

目的 研究耐甲氧西林金黄色葡萄球菌(MRSA)的内在化过程及内在化后引起宿主细胞的变化情况，并探讨宿主细胞[Ca2 ]i变化与MRSA内在化及细胞凋亡之间的关系。方法 利用激光扫描共聚焦显微镜追踪宿主细胞[Ca^2 ]i的动态变化，流式细胞术观察细胞凋亡情况。结果 MBSA ATCC-67-O不能被Hep2细胞内在化，却可以进入ECV304．细胞，并导致ECV304细胞凋亡，抑制细胞[Ca^2 ]i的升高未能影响内在化的菌数和凋亡的细胞数。结论 内在化的MBSA ATCC-67-O能够诱导FLV304．细胞发生凋亡。宿主细胞[Ca2 ]i变化与MBSA ATCC-67-O内在化及细胞凋亡之间并非直接的因果关系。     

骨髓基质细胞分泌蛋白的蛋白质质谱分析

目的通过Shotgun蛋白质组学分析,初步检测骨髓基质细胞(BMSCs)条件液中可能含有的蛋白质。方法将BMSCs条件液混匀后经超滤浓缩分为大于5kD和小于5kD两部分,并培养神经干细胞(NSCs),观察其后代中神经元、星形胶质细胞和少突胶质细胞的比例,鉴定出能调节NSCs分化的部分,并进行Shotgun蛋白质组学分析。结果BMSCs条件液分子量大于5kD的部分可以调节NSCs向神经元和少突胶质细胞方向分化,这部分条件液先经过聚丙烯酰胺凝胶电泳(SDS-PAGE)发现大部分蛋白集中在14kD以上,将蛋白条带酶解以后,经Shotgun分析共鉴定到456种蛋白质,其中154个相似蛋白质,17个假想蛋白质,56个未知蛋白质,剩余的229个蛋白质中大多为细胞骨架蛋白、分泌蛋白、信号转导蛋白、酶类和转运蛋白等。结论BMSCs分泌的多种蛋白质对NSCs的分化起调节作用,明确了BMSCs条件液中可能含有的成分。     

Characterization of an unusual cold shock protein from Staphylococcus aureus

Of the three cold shock proteins expressed by Staphylococcus aureus, CspC is induced poorly by cold but strongly by various antibiotics and toxic chemicals. Using a purified CspC, here we demonstrate that it exists as a monomer in solution, possesses primarily β‐sheets, and bears substantial structural similarity with other bacterial Csps. Aggregation of CspC was initiated rapidly at temperatures above 40 °C, whereas, the Gibbs free energy of stabilization of CspC at 0 M GdmCl was estimated to be +1.6 kcal mol^–1, indicating a less stable protein. Surprisingly, CspC showed stable binding with ssDNA carrying a stretch of more than three thymine bases and binding with such ssDNA had not only stabilized CspC against proteolytic degradation but also quenched the fluorescence intensity from its exposed Trp residue. Analysis of quenching data indicates that each CspC molecule binds with ～5 contiguous thymine bases of the above ssDNA and binding is cooperative in nature. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The influence of adhesive and invasive properties of Staphylococcus aureus defective in fibronectin-binding proteins on secretion of interleukin-6 by human endothelial cells

Fibronectin-binding proteins (FnBP) are surface adhesins of Staphylococcus aureus documented to be virulence attributes in, for example, endovascular infections. By using mutants of S. aureus defective in the FnBPA and B genes we have investigated whether these adhesins affect cytokine expression in human umbilical vein endothelial cells (HUVEC). S. aureus expressing FnBPA and B adhered to and were internalized into HUVEC to a greater extent compared to mutants defective in expression of FnBP. Production and release of IL-6 was higher from endothelial cells infected with the parent FnBP-expressing strain compared to the FnBP-defective mutants. These results indicate that adhesion to and invasion of S. aureus into endothelial cells are important regulators of cytokine expression.     

Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes

Small colony variants (SCVs) of Staphylococcus aureus have been proposed to persist within vascular endothelium, thereby sustaining chronic infections. To identify the intracellular SCV location we infected primary human endothelial cells with various wild type and SCV strains of S. aureus and visualised maturation of phagosomes using live cell imaging. Staphylococci-containing phagosomes were matured by sequential and dynamic interactions with Rab5- and Rab7-positive vesicles. Within 45–60 min all internalised staphylococci accumulated in LAMP-1- and LysoTracker-enriched lysosomal organelles and remained there for up to 5 days. Recovery of most staphyloccocal strains was below 1% after a 24 h intracellular stay, indicating a high bactericidal activity of the endothelial cell lysosomes. However, the menadione auxotroph SCV strain JB1 displayed a recovery rate of 4% and, furthermore, through multiple intracellular passaging a subtype (JB1-P4) with a recovery rate of 25–30% could be generated. Interestingly, both JB1 and JB1-P4 also resided exclusively in lysosomes. Thus, on one hand we document effective bactericidal activity of human endothelial cell lysosomes towards staphylococci, and on the other hand we provide evidence that certain SCVs are capable to withstand this activity.     

激活素A和骨形成蛋白-4诱导人羊膜上皮细胞转分化为心肌样细胞的实验研究

近年来羊膜上皮细胞(AECs)被认为是心肌细胞移植较好的种子细胞之一。研究表明,激活素A(ActivinA)和骨形成蛋白-4(BMP-4)能够诱导人胚胎干细胞向心肌细胞分化。本研究通过体外分离、培养并鉴定人AEC后,采用Activin A和BMP-4作为诱导剂,观察它们能否诱导AEC向心肌样细胞转分化。应用定量RT-PCR、Western blot、免疫细胞化学等方法来检测基因及蛋白表达变化。结果显示,人AEC能够表达上皮细胞特异性蛋白角蛋白19(CK19)。经Activin A和BMP-4共同诱导培养14d后,AEC心肌特异性转录因子Nkx2.5和特异性结构蛋白α-actinin的mRNA表达水平明显升高,同时α-actinin的蛋白表达水平亦明显增加。本实验提示,ActivinA和BMP-4在体外能够诱导人AEC向心肌样细胞转分化,同时该诱导方法可能成为诱导AEC向心肌样细胞转分化的一个新方法。     

Cross-reaction of anti-DNA autoantibodies with membrane proteins of human glomerular mesangial cells in sera from patients with lupus nephritis

Anti-DNA autoantibodies were thought to play a major role in the pathogenesis of lupus nephritis (LN). A recent study revealed that affinity-purified anti-DNA antibodies had a cross-reaction with human glomerular mesangial cells (HMC). However, whether the cross-reaction was antigen-antibody-mediated was unclear. The aim of the current study was to investigate the binding of anti-DNA antibodies to HMC membrane proteins and to characterize the target antigens. Affinity-purified IgG anti-DNA antibodies were purified by DNA-cellulose chromatography in sera from nine patients with biopsy-proven active lupus nephritis. In vitro cultured primary HMCs were disrupted by sonication and HMC membranes were obtained by differential centrifugation. The membranes of human umbilical vein endothelial cells (HUVEC), human proximal renal tubular epithelial cell line (HK2) and peripheral mononuclear cells (PMC) were obtained as controls. Binding of anti-DNA antibodies to the membrane proteins was investigated by Western blot analysis using soluble membrane proteins as antigens. Both HMC membrane and affinity-purified anti-DNA antibodies were treated with DNase I to exclude DNA bridging. All nine affinity-purified anti-DNA antibodies could blot the HMC membrane proteins, and there were at least three bands at 74 kDa, 63 kDa and 42 kDa that could be blotted. Among the nine IgG preparations, all nine (100%) could blot the 74 kDa band; eight (88.9%) could recognize 63 kDa and 42 kDa protein bands separately. After DNase treatment, the same bands could still be blotted by most affinity-purified anti-DNA antibodies. Affinity-purified anti-DNA antibodies could also blot similar bands on membrane proteins of other cells, but some bands were different. In conclusion, anti-DNA autoantibodies could cross-react directly with cell membrane proteins of human glomerular mesangial cells and might play an important role in the pathogenetic mechanism in lupus nephritis.     

Characterization of epithelial V-like antigen in human choroid plexus epithelial cells: potential role in CNS immune surveillance

Near-infrared spectroscopy (NIRS) can detect two different kinds of signals from the human brain: the hemodynamic response (slow) and the neuronal response (fast). This paper explores a nonlinear aspect in the tactile-stimulus-evoked neuronal optical response over a NIRS time series (light intensity variation). The existence of the fast optical responses (FORs) over the time series recorded in stimulus sessions is confirmed by event-related averaging. The chaos levels of the NIRS time series recorded both in stimulus and in rest sessions are then identified according to the estimated largest Lyapunov exponent. The obtained results ascertain that stimulus-evoked neuronal optical responses can be detected in the somatosensory cortex using continuous-wave NIRS equipment. Further, the results strongly suggest that the chaos level can be used to recognize the FORs in NIRS time series and, thereby, the state of the pertinent brain activity.     

c-myc基因在EBV转化人胃上皮细胞中的作用

目的研究EBV感染人胃上皮细胞系GES-1后c-myc基因的表达状况及转化作用,以探讨c-myc基因在Epstein-Barr病毒(EBV)相关胃癌发生中所起的作用。方法用携带NEO~r基因的重组EBV产生细胞系Akata 1061以密切接触法感染人胃上皮细胞系GES-1,采用脂质体介导法将c-myc基因转染至同一细胞,以pcDNA3空载体质粒转染同一细胞为对照;经G418筛选获得感染或转染的抗性细胞克隆;采用免疫细胞化学法测定EBNA1的表达以鉴定EBV感染细胞克隆,测定EBV感染和c-myc基因转染细胞中C-myc蛋白的表达;通过形态学观察、细胞生长曲线测定及流式细胞分析等方法观察细胞生物学特性的变化。结果与对照组相比,EBV感染及c-myc转染后细胞发生明显的形态学变化,生长速度明显加快,S期细胞比例显著提高,分别为(70.96±0.91)%和(60.67±3.06)%vs(34.74±1.03)%,P<0.05,表明EBV感染及c-myc基因转染均使细胞增殖加快。结论EBV感染及c-myc基因转染可使人胃上皮细胞的增殖速度加快,细胞恶性程度增强,但并未导致肿瘤的发生。EBV对人胃上皮细胞的转化作用可能与c-myc基因的过度表达有关。     

高糖预处理影响单核细胞对热灭活金黄色葡萄球菌刺激的反应

目的 观察高糖状态下热灭活金黄色葡萄球菌(HKSA)诱导的人单核细胞株THP-1凋亡特点,以及表达诱导型一氧化氮合酶(iNOS)、白细胞介素(IL)-1β mRNA和IL-1β蛋白分泌规律.方法 体外培养THP-1单核细胞,分别以5.5 mmol/L葡萄糖(LG)和25.0 mmol/L葡萄糖(HG)培养12h～8d.将HG和LG培养6d的单核细胞加入HKSA.分别于HKSA加入前和加入后2～48 h提取单核细胞,检测细胞凋亡、IL-1β蛋白、iNOS和IL-1β mRNA表达.细胞凋亡采用Annexin V与PI双染法,流式细胞技术检测;IL-1β蛋白分泌采用ELISA法检测;提取细胞总RNA,反转录生成cDNA后采用实时定量逆转录聚合酶链反应(real-time quantitative PCR)法,检测iNOS和IL-1β mRNA表达情况.结果 (1)高糖刺激THP-1单核细胞12 ～48 h后IL-1β蛋白、iNOS和IL-1β mRNA表达较刺激前显著增加(P＜0.05),iNOS和IL-1β mRNA表达在24 h达最高值,IL-1β蛋白分泌48 h达高峰.细胞凋亡在高糖刺激48 h～4 d较刺激前显著增加(P＜0.05).(2)两组细胞加入HKSA后6～48 h表达iNOS和L-1β显著高于刺激前(P＜0.01),24h达高峰,48 h表达下降;IL-1β蛋白分泌在48 h达最高值.两组细胞凋亡率在加入HKSA后12h、24h、48 h逐渐增加(P＜0.01).单核细胞加入HKSA前和加入HKSA后12h,高糖组与低糖组比较,高糖组IL-1β蛋白、iNOS和IL-1β mRNA表达均低于低糖组,凋亡率高于低糖组(P＜0.05).结论 高糖和HKSA对单核细胞分泌IL-1β蛋白、表达iNOS和IL-1β mRNA的影响与刺激时间有关;单核细胞处于持续高糖状态时,高糖可以增加细胞凋亡率,降低IL-1β蛋白分泌、iNOS和IL-1β表达.     

The human severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesis.     

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330₂₃₃ primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood. J. Med. Virol. 53:81–84, 1997. © 1997 Wiley-Liss, Inc.     

人上皮性卵巢癌细胞系侧群细胞的肿瘤干细胞样细胞生物学特性及膜蛋白差异表达的鉴定

目的 鉴定上皮性卵巢癌细胞侧群细胞(SP细胞)肿瘤干细胞样细胞生物学特性,并检测侧群细胞内差异蛋白质的表达.方法 流式分选上皮性卵巢癌细胞系SKOV3和A2780具有外泌Hochest33342染料生物学特性的侧群细胞,鉴定其肿瘤干细胞样细胞相关生物学特性.运用细胞培养稳定同位素标记(SILAC)的定量蛋白质组学技术,...     

Polarized M2c macrophages have a promoting effect on the epithelial-to-mesenchymal transition of human renal tubular epithelial cells

This study planned to explore the effects of M2c macrophages on epithelial-to-mesenchymal transition (EMT) of human renal proximal tubular epithelial cells (HK-2). Human monocytic leukaemia cells were induced by TPA and IL-10 to differentiate M2c macrophages. Subsequently HK-2 cells were co-cultured with the M2c macrophages in Transwell chamber. After 48?h of co-culturing the HK-2 cells were detected in the mRNA and protein expression of E-cadherin, α-SMA and vimentin with RT-PCR, immunofluorescence and Western blot respectively. Besides, the migration ability of the HK-2 cells was estimated with Transwell migration assay. ANOVA was used to compare the difference between groups and Student's t-test to conduct multiple comparisons of two groups. P?<?0.05 was considered statistically significant. The results showed that the mRNA and protein expression of α-SMA and vimentin of the HK-2 cells were increased but the E-cadherin decreased significantly after 48?h co-culturing with the M2c macrophages (P?<?0.05 or P?<?0.01). And the migration ability of HK-2 cells were also increased significantly (P?<?0.05). It may be concluded that polarized M2c macrophages may have a promoting effect on the EMT of HK-2 cells.     

Innate response of human endothelial cells infected with mycobacteria

Endothelial cells are susceptible to infection by several pathogens, but little is known about mycobacterial infection. We analyzed some features of mycobacteria-endothelial cell interactions and the innate response to the infection. Intracellular growth in human umbilical vein endothelial cells (HUVECs) of three Mycobacterium species: M. tuberculosis (MTB), M. abscessus (MAB) and M. smegmatis (MSM) was analyzed. M. smegmatis was eliminated; M. abscessus had an accelerate intracellular replication and M. tuberculosis did not replicate or was eliminated. M. abscessus infection induced profound cytoskeleton rearrangements, with M. tuberculosis infection changes were less marked, and with MSM were slight. Nitric oxide (NO) production was induced differentially: M. abscessus induced the highest levels followed by M. tuberculosis and M. smegmatis; the contrary was true for reactive oxygen species (ROS) production. Only M. tuberculosis infection caused beta-1 defensin over-expression. As a whole, our results describe some aspects of the innate response of HUVEC infected by mycobacteria with different virulence and suggest that a strong cytoskeleton mobilization triggers a high NO production in these cells.     

The vaccinia virus fusion inhibitor proteins SPI-3 (K2) and HA (A56) expressed by infected cells reduce the entry of superinfecting virus

The orthopoxvirus SPI-3 (K2) and A56 (hemagglutinin, HA) proteins interact and together prevent cell–cell fusion. SPI-3/A56 has been proposed to prevent the superinfection of previously infected cells by reducing virus–cell fusion. Binding of mature virions of vaccinia virus (VV) to VV-infected cells was unaffected by SPI-3 or A56 on the surface of infected cells. Entry of VV into infected cells was assessed using VV-P_T7-luc carrying the luciferase reporter under T7 control. Cells infected with VV or cowpox virus (CPV) expressing T7 RNA polymerase and lacking SPI-3 and/or A56 were superinfected with VV-P_T7-luc, and luciferase activity was measured. Inactivation of SPI-3 or A56 from the pre-infecting virus resulted in greater luciferase expression from the superinfecting VV-P_T7-luc. Antibody against SPI-3 present during infection with wild-type CPV-T7 increased luciferase expression from superinfecting VV-P_T7-luc. The SPI-3/A56 complex on the infected cell surface therefore appears to reduce the entry of virions into infected cells.     

Polarization fluorescence, spin label and ultracentrifugal studies of specific interaction of low molecular weight proteins with the Fc fragment of human immunoglobulin G

Low mol. wt (about 2000) proteins which were found in normal human serum formed complexes with the Fc fragment of IgG. The interaction constant was not less than 10⁶ 1/mole. These complexes dissociated on dilution of the protein solution to below 2 μM or decreasing the pH below 6. The binding site of these proteins was located in the C_H2 domains in close proximity to the carbohydrate moieties. The dissociation of IgG complexes with these proteins was accompanied by a conformational change in the Fc fragment.