可溶性CD40配体的表达及其对树突状细胞和恶性B细胞的影响(英文)

CD4 0配体 (CD4 0L)是TNF超家族成员 ,表达于人活化的CD4 T细胞表面 ,与存在于B细胞、抗原递呈细胞表面的相应受体CD4 0分子相互作用 ,促发并调节机体的体液免疫和细胞免疫应答。用PCR法从CD4 0L全长cDNA质粒中扩增CD4 0L胞外段编码cDNA ,即可溶性CD4 0L(sCD4 0L)编码cDNA ,并克隆入 pSK质粒 ;cDNA序列经测序证实后与 pPICZαA质粒重组构建成 pPICZαA sCD4 0L分泌型表达载体并在GS1 1 5毕氏酵母菌株中成功地表达出人可溶性CD4 0配体(rhsCD4 0L) ,最高表达量为 35mg/L(培养上清 )。结果显示 :在无TNFα存在时 ,重组人可溶性CD4 0L(rhsCD4 0L)可有效地诱导人外周单核细胞向树突状细胞 (DC)的分化 ,具有典型的DC形态特征和特殊表面标志 (CD1a、CD80、CD83等 ) ,为DC在体外的大量扩增提供新手段 ;进一步研究发现 ,rhsCD4 0L能直接并显著地抑制恶性淋巴瘤细胞株Daudi和多发性骨髓瘤细胞株XG 2的体外增殖 ;并证实rhsCD4 0L与CD4 0L基因转染细胞 (L/CD4 0L TC)一样 ,在体外均诱导XG2细胞的凋亡 ;rhsCD4 0L有希望成为一种重要的免疫调节剂和新的抗肿瘤药物     

Evaluation of the Potential In Vitro Antiproliferative Effects of Millimeter Waves at Some Therapeutic Frequencies on RPMI 7932 Human Skin Malignant Melanoma Cells

The potential antiproliferative effects of low power millimeter waves (MMWs) at 42.20 and 53.57 GHz on RPMI 7932 human skin melanoma cells were evaluated in vitro in order to ascertain if these two frequencies, comprised in the range of frequency used in millimeter wave therapy, would have a similar effect when applied in vivo to malignant melanoma tumours. Cells were exposed for 1 h exposure/day and to repeated exposure up to a total of four treatments. Plane wave incident power densities <1 mW/cm² were used in the MMWs-exposure experiments so that the radiations did not cause significant thermal effects. Numerical simulations of Petri dish reflectivity were made using the equations for the reflection coefficient of a multilayered system. Such analysis showed that the power densities transmitted into the aqueous samples were ≤0.3 mW/cm². Two very important and general biological endpoints were evaluated in order to study the response of melanoma cells to these radiations, i.e. cell proliferation and cell cycle. Herein, we show that neither cell doubling time nor the cell cycle of RPMI 7932 cells was affected by the frequency of the GHz radiation and duration of the exposure, in the conditions above reported.     

靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响

目的 构建携带促血管生成素2-小干扰RNA（Ang2-siRNA）慢病毒载体,观察其对恶性黑色素瘤细胞中Ang2基因表达的干扰作用.方法 将经XbaⅠ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒（pNL-EGFP）载体与pSilencer 1.0-U6启动子-促血管生成素2-小干扰RNA（pSilencer 1.0-U6-Ang2-siRNA）重组质粒连接,产生加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅰ（pNL-EGFP-U6-Ang2-Ⅰ）、加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅱ（pNL-EGFP-U6-Ang2-Ⅱ）慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒、水疱性口炎病毒G蛋白（pVSVG）包膜质粒和pHelper包装质粒共转染293T细胞,产生pNL-EGFP-U6-Ang2-Ⅰ、pNL-EGFP-U6-Ang2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Ang2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Ang2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.0×103/ml.实时荧光定量RT-PCR结果显示：Ang2-siRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Ang2基因的表达（P＜0.05）.结论 成功构建了Ang2-SiRNA慢病毒载体,体外研究显示Ang2-SiRNA慢病毒载体能抑制恶性黑色素瘤细胞中Ang2 mRNA的表达,为下一步进行裸鼠恶性黑色素瘤移植瘤生长的干预实验奠定基础,为肿瘤的基因治疗提供实验依据.     

Sensitive Immunofluorescent Staining of Cells via Generation of Fluorescent Nanoscale Polymer Films in Response to Biorecognition

Immunofluorescent staining is central to nearly all cell-based research, yet only a few fluorescent signal amplification approaches for cell staining exist, each with distinct limitations. Here, the authors present a novel, fluorescent polymerization-based amplification (FPBA) method that is shown to enable similar signal intensities as the highly sensitive, enzyme-based tyramide signal amplification (TSA) approach. Being non-enzymatic, FPBA is not expected to suffer from nonspecific staining of endogenous enzymes, as occurs with enzyme-based approaches. FPBA employs probes labeled with photopolymerization initiators, which lead to the controlled formation of fluorescent polymer films only at targeted biorecognition sites. Nuclear pore complex proteins (NPCs; in membranes), vimentin (in filaments), and von Willebrand factor (in granules) were all successfully immunostained by FPBA. Also, FPBA was demonstrated to be capable of multicolor immunostaining of multiple antigens. To assess relative sensitivity, decreasing concentrations of anti-NPC antibody were used, indicating that both FPBA and TSA stained NPC down to a 1:100,000 dilution. Nonspecific, cytoplasmic signal resulting from NPC staining was found to be reduced up to 5.5-fold in FPBA as compared to TSA, demonstrating better signal localization with FPBA. FPBA’s unique approach affords a combination of preferred attributes, including high sensitivity and specificity not otherwise available with current techniques.     

The reaction of Acridine Orange with proteoglycans in the articular cartilage of the rat

Summary Acridine Orange in concentrations from 0.01% to 0.2% was added to the first fixative solution in order to stain vibratome sections and small blocks of the articular cartilage of 2 month old rats. The interterritorial matrix of the radial or deep zone (zone 3) was examined. It contained reaction products with different morphology depending on the specimens used. In vibratome sections filaments were seen arranged in a homogenous pattern and changing in size with the concentration of the dye: diluted solutions produced finer filaments than concentrated ones. In contrast, in tissue blocks the staining pattern was not altered by different concentrations of Acridine Orange. However, with increase of the distance from the surface of the specimens the size of the filaments gradually decreased and formed a finer network. Since after preincubation with chondroitin ABC lyase only minute reaction products remained, an interaction of the dye with the sulphated glycosaminoglycans of the proteoglycans in the articular cartilage is suggested.The experiments show that by using mainly monocationic monomers of Acridine Orange the proteoglycans can be stained in a more expanded state than with polycationic dye polymers.     

Ad-GFP-nm23-H1裸鼠体内抑制人恶性黑色素瘤A375作用的研究

目的探讨Ad—GFP—nm23-H1对人恶性黑素瘤裸鼠皮下移植瘤的抑制作用,从而为后期nm23-H1基因和腺病毒载体用于人恶性黑色素瘤及其它肿瘤的基因治疗提供一定的理论和方法。方法在裸鼠真皮下建立人A375细胞黑色素瘤动物模型后,设对照组及10^9 PFU／ml、10^10 PFU／ml的Ad—GFP—nm23-H1干预组。经Ad—GFP—nm23-H1干预治疗后,取瘤体称重,计算抑瘤率,通过光镜进行瘤组织病理形态学观察。结果对照组及10^9 PFU／ml、10^10 PFU／ml的Ad—GFP-nm23-H1干预组的平均肿瘤体积和瘤重分别为：1．4129&#177;0．4832mm^3、1．1914&#177;0．3304mm^3、0．75&#177;0．2548mm^3和1．924&#177;0．539g、1．655&#177;0．5754g、1．195&#177;0．2639g。与另两组比较,10^10 PFU／ml的Ad．GFP—nm23-H1干预组对A375具有明显的抑制作用。结论10^10 PFU／ml的Ad—GFP—nm23-H1干预组对裸鼠移植A375实体瘤有明显的抑制作用。     

Multiplication of Legionella pneumophila in HeLa Cells in the Presence of Cytoskeleton and Metabolic Inhibitors

A study has been carried out on the action of cytoskeleton and metabolic inhibitors on intracellular multiplication in HeLa cells of a virulent strain of Legionella pneumophila serogroup 6. The effects of the substances were separately tested on both penetration and intracellular multiplication of L. pneumophila. Only cytochalasin A and 2-deoxy-d -glucose (2dG) affected bacterial internalisation, whereas intracellular multiplication was inhibited by cytochalasins A, B, C, D and J (D being the most active) and by 2dG with a dose-response effect. The action of 2dG was counteracted by 50 mM glucose. Experiments carried out with cytochalasin D and a rhodamine-phalloidin conjugate showed the involvement of cytoskeletal elements in intracellular multiplication of Legionella; compounds acting on microtubules had no effect.     

The Non-Coding RNA Llme23 Drives the Malignant Property of Human Melanoma Cells

Several lines of evidence support the notion that increased RNA-binding ability of polypyrimidine tract-binding (PTB) protein-associated splicing factor (PSF) and aberrant expression of long non-coding RNAs (lncRNAs) are associated with mouse and human tumors. To identify the PSF-binding lncRNA involved in human oncogenesis, we screened a nuclear RNA repertoire of human melanoma cell line, YUSAC, through RNA-SELEX affinity chromatography. A previously unreported lncRNA, termed as Llme23, was found to bind immobilized PSF resin. The specific binding of Llme23 to both recombinant and native PSF protein was confirmed in vitro and in vivo. The expression of PSF-binding Llme23 is exclusively detected in human melanoma lines. Knocking down Llme23 remarkably suppressed the malignant property of YUSAC cells, accompanied by the repressed expression of proto-oncogene Rab23. These results may indicate that Llme23 can function as an oncogenic RNA and directly associate the PSF-binding lncRNA with human melanoma.     

Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: role of TIMP-2 and plasma membranes

MMP-9 (gelatinase B) is produced in a latent form (pro-MMP-9) that requires activation to achieve catalytic activity. Previously, we showed that MMP-2 (gelatinase A) is an activator of pro-MMP-9 in solution. However, in cultured cells pro-MMP-9 remains in a latent form even in the presence of MMP-2. Since pro-MMP-2 is activated on the cell surface by MT1-MMP in a process that requires TIMP-2, we investigated the role of the MT1-MMP/MMP-2 axis and TIMPs in mediating pro-MMP-9 activation. Full pro-MMP-9 activation was accomplished via a cascade of zymogen activation initiated by MT1-MMP and mediated by MMP-2 in a process that is tightly regulated by TIMPs. We show that TIMP-2 by regulating pro-MMP-2 activation can also act as a positive regulator of pro-MMP-9 activation. Also, activation of pro-MMP-9 by MMP-2 or MMP-3 was more efficient in the presence of purified plasma membrane fractions than activation in a soluble phase or in live cells, suggesting that concentration of pro-MMP-9 in the pericellular space may favor activation and catalytic competence.     

The Role of Alpha-Synuclein in Melanin Synthesis in Melanoma and Dopaminergic Neuronal Cells

The relatively high co-occurrence of Parkinson’s disease (PD) and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR)-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM), the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn) that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR) and inhibit tyrosine hydroxylase (TH), both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA), led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB) light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in melanoma cells and in dopaminergic neuronal cells.     

Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K _m of 0.85 μM. The k _cat and k _cat?K _m values were 13 s^?1 and 15 s^?1 μM^?1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K _i, of 25 pM.     

Double Fluorescent Staining for the Separate Demonstration of Chromosomes and Microtubules in Mitotic Cells In Vitro

A simple fluorescent method for double staining of mitotic cells using a rhodamine B indirect immunofluorescent method for tubulin and the DNA-specific fluorescent dye Hoechst 33258 for nuclei and chromosomes is described. This procedure enables one through the use of appropriate excitation filters to view at will either chromosomes and nuclei or tubulin within the same cell.     

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P₁ (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P′₂ favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P₁ and P′₂ substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.     

Genistein Enhances the Cisplatin‐Induced Inhibition of Cell Growth and Apoptosis in Human Malignant Melanoma Cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy‐refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μM) did not induce apoptosis by itself but did enhance cisplatin‐induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti‐apoptotic proteins, bcl‐2 and bcl‐xL, and one pro‐apoptotic protein, apoptotic protease activating factor‐1 (Apaf‐1), were examined. Genistein alone or cisplatin alone generally did not alter bcl‐2 expression or bcl‐xL expression, but slightly increased Apaf‐1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl‐2 and bcl‐xL protein and increased Apaf‐1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

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The Effect of Paraquat on the Radiosensitivity of Melanoma Cells: the Role of Superoxide Dismutase & CATALASE

The activities of reactive oxygen species scavenging enzymes, superoxide dismutases (SODs) and catalase (in cells of two melanomas (mouse B16 and human SK23) and in Chinese hamster ovary (CHO) cells were examined. Melanoma cells are relatively depleted in activities of superoxide dismutases and catalase as compared to CHO cells. Short equitoxic (500μM for CHO and B16 cells and 5 μM for SK23 cells) paraquat treatment (15 min before the X-irradiation, 45 min in postirradiation period - the total time of treatment was 1 h) caused an increase in radiation resistance, measured as colony forming ability, in two of the three lines examined.

It is proposed that PQ may exert its radioprotective effect by induction of antioxidant enzymes.