Mouse Dendritic Cell (DC) Influenza Virus Infectivity Is Much Lower than That for Human DCs and Is Hemagglutinin Subtype Dependent

We show that influenza A H1N1 virus infection leads to very low infectivity in mouse dendritic cells (DCs) in vitro compared with that in human DCs. This holds when H3 or H5 replaces H1 in recombinant viruses. Viruslike particles confirm the difference between mouse and human, suggesting that reduced virus entry contributes to lower mouse DC infectivity. Low infectivity of mouse DCs should be considered when they are used to study responses of DCs that are actually infected.     

Novel Cationic Phosphonolipids Agents for in Vitro Gene Transfer to Epithelial Cell Lines

Abstract

The use of cationic liposomes is one of the main approaches currently investigated to introduce into a cell a gene with therapeutic properties. This study presents in vitro results obtained with a new family of gene transfer agents, the phosphonolipids. We have synthesized 37 members of this family and optimized the conditions of in vitro gene transfer targeted at a lung epithelial cell line (CFT1 cells) by using a reporter gene (β-galactosidase). Two quantitative tests, a CPRG¹ (Chlorophenol red galactopyranoside) test and a Flow cytometric assay (FACS-Gal¹ assay), have been used to determine the percentage of transfected cells. The cationic phosphonolipids were tested alone or formulated with 50% DOPE¹ (Dioleoylphosphatidylethanolamine) (w:w). The results obtained with the CPRG test led us to select 7 compounds that were more efficient than the commercialized lipids Lipofectin, Lipofectamine and Transfectam. The transfer kinetics of the transgene were studied and showed that more than 20 % of cells were still positive at day 7 after transfection.     

Primary Treatment Response Rather than Front Line Stem Cell Transplantation Is Crucial for Long Term Outcome of Peripheral T-Cell Lymphomas

Outcome of systemic peripheral T-cell lymphomas (PTCL) is unsatisfactory and no controlled clinical study guides the therapy. Phase II studies suggest to consolidate response achieved after front-line treatment with stem cell transplant (SCT). We retrospectively evaluate the impact of front-line SCT consolidation in a single Center cohort of 209 patients treated during the last two decades. Median age was 49 years (range 15-85) with a prevalence of male sex (61%), advanced stage (68%) while IPI was >2 in 44%. Primary treatment was MACOP-B (39%) CHO(E)P (39%), intensive regimens (18%) or others (4%). Complete response to primary treatment (i.e. before SCT) was 60% (5% partial remission). Forty-four patients further proceeded to SCT while 92 did not receive consolidation. Outcome of primary responders was good, with a 3-year overall survival of 74% (82% in ALCL ALK+ and 69% for the other histologies). By multivariate analysis a better overall survival was significantly associated with IPI<2 (P=0.001), primary response (P=0.000), and ALCL ALK+ (P=0.012). The multivariate analysis performed on responders, showed that only IPI was predictive of a better survival while ALCL ALK+ and undergoing SCT were not. Response to primary treatment rather than post-remission programs is the crucial determinant of PTCL outcome.     

Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea

Quantitative real-time RT-PCR (qPCR) has been widely used to investigate gene expression during seed germination, a process involving seed transition from dry/physiologically inactive to hydrated/active state. This transition may result in altered expression of many housekeeping genes (HKGs), conventionally used as internal controls, thereby posing a challenge about selection of HKGs in such scenarios. The objectives of this study included identifying valid reference genes for seed priming and germination studies, both of which involve the transition of seed hydration status, and assessing whether or not findings derived from the “seed model” used in this study would also be applicable to other plant species. Eight commonly used HKGs were evaluated in maize seeds during hydropriming and germination. Using Bestkeeper, geNorm, and NormFinder, we provided a rank of stability for these HKGs. Actdf, UBQ, βtub, 18S, Act, and GAPDH were adjudged as valid internal controls by geNorm and NormFinder. Under the second objective, we conducted a case study with spinach seeds collected during osmopriming and germination. Our results indicate that the conclusions derived from maize were applicable to spinach as well, in that 18S exhibited greater expression stability than GAPDH in osmoprimed and germinated seeds; this held true even under stress conditions. While both of these genes were rejected by BestKeeper, we found that 18S exhibited stable expression when “dry” and “hydrated” seeds were analyzed as separate data sets. Although this approach precludes the comparison between “hydrated” and “dry” seeds, it still provides effective comparison among samples of same hydration status.     

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples.     

in Vitro Effect of Deoxynivalenol on the Differentiation of Human Colonic Cell Lines Caco-2 and t84

The effects of low concentrations of deoxynivalenol (DON) on structural and functional characteristics of human colonic adenocarcinoma cell lines Caco-2 and T84 were examined. Scanning electron microscopic (SEM) analysis of the apical surfaces of Caco-2 cells revealed reduction or abnormal formation of brush borders in the presence of 50, 100 and 200 ng/ml of DON. Monolayer integrity of Caco-2 and T84 cells was studied using cells which were cultured on permeable membranes. The transepithelial electrical resistance (TEER) of Caco-2 cells was significantly reduced at 50, 100 and 200 ng/ml of DON, significant increase in lucifer yellow (LY) permeability was also observed in these cells at 100 ng/ml of DON. The TEER of T84 cells was significantly reduced at 100 and 200 ng/ml of DON. LY permeability significantly increased at 200 ng/ml of DON in T84 cells. Enzyme activities in Caco-2 cells were also examined. Alkaline phosphatase activity was reduced from the 6th to 15th day of culture in the presense of 100 or 200 ng/ml of DON, whereas sucrase- isomaltase activity was significantly decreased by adding 50 or 100 ng/ml of DON for 15 or 20 days. Protein content was attenuated only by treatment with 200 ng/ml of DON thoughout the experimental period. The results indicate that DON interferes with structural and functional characteristics of differentiation in enterocytes at low doses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination and Validation of Reference Gene Stability for qPCR Analysis in Polysaccharide Hydrogel-Based 3D Chondrocytes and Mesenchymal Stem Cell Cultural Models

Gene expression study is widely used to obtain information of the cell activities and phenotypes. To quantify gene expression, measurement of the mRNA copy number is commonly done by quantitative RT-PCR (RT-qPCR). However, proper reference gene is needed for different tissues to normalize the expression level of different genes accurately. In this study, reference gene determination was done for three-dimensional (3D) artificial tissue constructs in hydrogel. Porcine synovium-derived mesenchymal stem cells (SMSCs) and rabbit chondrocytes were cultured in both alginate and agarose hydrogels to set up four different 3D culture systems to form the artificial tissue constructs. The gene expression levels of candidate genes were determined by RT-qPCR and then analyzed by geNorm, Bestkeeper, and Normfinder. For porcine SMSCs, PPIA, and TBP were selected for tissue in alginate scaffold whereas HPRT and TBP were selected for the agarose scaffold system. On the other hand, HPRT, PPIA, and RPL18 were the stable reference genes for rabbit chondrocytes in alginate scaffold while TBP, RPL5, and RPL18 were selected for rabbit chondrocytes in agarose scaffold. This study has further indicated that suitable reference genes are different for each tissue and study purpose. The reference genes are expressed in different stability when a scaffold of different material is used.     

腺病毒5型E1A基因对永生化人支气管上皮细胞生长的抑制作用及其机理

腺病毒 5型早期区 1 A( Ad5E1 A)基因是新近发现的一个肿瘤抑制基因 .其产物 E1 A蛋白是多功能转录因子 ,它能从正、负 2个途径调控多种细胞基因的转录 ,具有降低体内致瘤性及抗转移等活性 .为了探讨 E1 A基因对代表肺癌癌前病变的永生化人支气管上皮细胞的生长是否具有抑制作用 ,构建了在真核细胞高表达 E1 A基因的重组质粒 p CEP4- E1 A.通过脂质体介导将 E1 A基因转入永生化人支气管上皮细胞第 1 68代 ( MP1 68)中 ,经潮霉素筛选 ,获得稳定表达 E1 A的永生化人支气管上皮细胞 ( MP1 68- E1 A) .结果表明 :E1 A基因的稳定表达抑制了 HER- 2 / neu基因的表达 .转染细胞 ( MP1 68- E1 A)回复扁平形态、恢复细胞生长的接触性抑制 ,细胞群体生长缓慢 (倍增时间是 MP1 68- vect细胞的 1 .41倍 ) ,细胞周期 G1期阻滞并出现凋亡 ,软琼脂集落形成抑制率达73.86% .结果说明 E1 A基因的稳定表达明显抑制了永生化人支气管上皮细胞的生长 .该作用可能与 E1 A抑制 HER- 2 / neu基因的表达及诱导永生化人支气管上皮细胞凋亡有关 .     

Eupatorin-Induced Cell Death in Human Leukemia Cells Is Dependent on Caspases and Activates the Mitogen-Activated Protein Kinase Pathway

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G₂-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation.     

Select de novo Gene and Protein Expression During Renal Epithelial Cell Culture in Rotating Wall Vessels is Shear Stress Dependent

The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types. Received: 30 June 1998/Revised: 30 November 1998     

DNA Hypermethylation and Partial Gene Silencing of Human Thymine-DNA Glycosylase in Multiple Myeloma Cell Lines

Multiple myeloma (MM) has prominent features of karyotypic instability at the earliest stage, leading to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways. A possible mechanism of dysregulation of the DNA repair pathway is epigenetic gene silencing. Therefore, we sought to determine the methylation status of enzymes involved in the base excision repair pathway in multiple myeloma cell lines. Here, we report the aberrant DNA methylation of TDG, one of the enzymes involved in base excision repair of damaged DNA, in several multiple myeloma cell lines but not in normal human plasma cells. DNA hypermethylation of TDG in the MM cell lines leads to lower gene expression levels that results in less efficient DNA repair activity in response to hydrogen peroxide-induced DNA damage. Expression of exogenous TDG can functionally compensate for lower repair activities of damaged DNA in the KAS-6/1 myeloma cell line, which has extensive DNA hypermethylation of the TDG promoter. Hypermethylation of DNA damage repair genes in MM cell lines may provide an explanation for the frequent genomic instability, as well as point mutations, that are encountered in MM.     

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.Internalization of epidermal growth factor receptors (EGFR)² and their subsequent delivery to lysosomes play key roles in attenuating EGF-mediated signaling cascades (1, 2). The proper delivery of EGFR into lysosomes for degradation requires a series of highly regulated targeting and delivery events. Following ligand binding, EGFR is internalized via endocytic vesicles that are subsequently targeted to early endosomes. This targeting event is mediated by the small GTPase, Rab5 (3, 4). Once delivered to the early endosome, receptors that are destined for degradation are incorporated into vesicles that bud into the lumen of the endosome, forming the multivesicular body (reviewed in Refs. 5, 6). Sequestration of the activated cytoplasmic domain of EGFR into the intralumenal vesicles of the multivesicular body effectively terminates receptor signaling (7). Subsequent fusion of the multivesicular body with lysosomes delivers the intralumenal vesicles and their contents into the lumen of the lysosome where they are degraded (reviewed in Refs. 8–10). Inactivating mutations in Rab5 disrupt the delivery of cell surface receptors, such as EGFR, to early endosomes, thereby inhibiting receptor trafficking to the lysosome and receptor degradation (11, 12). Therefore, activation of Rab5 is a key point of regulation for EGFR signaling.Rab5 cycles between an inactive GDP-bound state and an active GTP-bound state, and Rab5 activation requires the exchange of GDP to GTP. This exchange is catalyzed by guanine nucleotide exchange factors (GEFs) that are specific to the Rab5 family of proteins (reviewed in Ref. 13). Rab5 family GEFs all contain a catalytic vacuolar protein sorting 9 (Vps9) domain that facilitates the GDP to GTP exchange (14–17). Many Rab5 GEFs contain other functional domains that are involved in cell signaling events (13). Rin1 is a good example of a multidomain Rab5 GEF. In addition to the Vps9 domain, Rin1 also contains an Src homology 2 domain, a proline-rich domain, and a Ras association domain. Rin1 was originally identified through its ability to interact with active Ras (18), and a role for Rin1 in a number of cell signaling systems has been established, including EGF-mediated signaling (19–21). Rin1 directly interacts with the activated EGFR through its Src homology 2 domain (22). Furthermore, Ras occupation of the Rin1 Ras association domain positively impacts the Rab5 GEF activity of Rin1, which promotes EGFR internalization and attenuation in fibroblasts (23). However, Rin1 expression is up-regulated in several types of cancers, including squamous cell carcinoma (24), colorectal cancer (25), and cervical cancer (26), through duplications or rearrangements of the RIN1 locus. These studies suggest that Rin1 may also play a role in enhancing cell proliferation.It is well established that a large percentage of non-small cell lung adenocarcinomas exhibit up-regulation of EGFR and aberrant signaling through the Ras/MAPK pathway (reviewed in Ref. 27). In addition, a recent study examining 188 human lung adenocarcinomas identified that 132 of 188 tumor samples exhibited mutations relating to the Ras/MAPK signaling pathway (28). Accordingly, the role of Rin1 in non-small cell lung adenocarcinoma was addressed. Examination of a panel of non-small cell lung adenocarcinoma lines (including A549) revealed enhanced Rin1 expression relative to a nontransformed lung epithelial cell line (BEAS-2B). Depletion of Rin1 from A549 cells resulted in decreased proliferation. This decrease correlated with a reduction in EGF-activated ERK phosphorylation and the stabilization of cell surface EGFR. These defects were complemented by wild type Rin1 expression but not by mutant Rin1 lacking a functional Vps9 domain, suggesting that the GEF activity of Rin1 is necessary for proper EGFR signaling in A549 cells. In addition, overexpression of Rin1Δ, dominant negative Rab5, and dynamin resulted in similar defects in cell proliferation and EGFR signaling as Rin1 depletion. These data indicate that proper EGFR internalization and trafficking are critical for robust EGFR-mediated signaling and cell proliferation in A549 cells and offer evidence that Rin1 positively regulates cell proliferation in non-small cell lung adenocarcinoma.     

Human Immunodeficiency Virus Type 1 Attachment to HeLa CD4 Cells Is CD4 Independent and gp120 Dependent and Requires Cell Surface Heparans

The binding of human immunodeficiency virus type 1 (HIV-1) (Hx10) virions to two different cell lines was analyzed by using a novel assay based on the detection, by anti-HLA-DR-specific antibodies, of HLA-DR⁺ virus binding to HLA-DR⁻ cells. Virion attachment to the CD4⁺-T-cell line A3.01 was highly CD4 dependent in that it was potently inhibited by CD4 monoclonal antibodies (MAbs), and little virus binding to the CD4⁻ sister A2.01 line was observed. By contrast, virion binding to HeLa cells expressing moderate or high levels of CD4 was equivalent to, or lower than, binding to wild-type CD4⁻ HeLa cells. Moreover, several CD4 MAbs did not reduce, but enhanced, HIV-1 attachment to HeLa-CD4 cells. CD4 was required for infection of HeLa cells, however, demonstrating a postattachment role for this receptor. MAbs specific for the V2 and V3 loops and the CD4i epitope of gp120 strongly inhibited virion binding to HeLa-CD4 cells, whereas MAbs specific for the CD4bs and the 2G12 epitopes enhanced attachment. Despite this, all gp120- and gp41-specific MAbs tested neutralized infectivity on HeLa-CD4 cells. HIV-1 attachment to HeLa cells was only partially inhibited by MAbs specific for adhesion molecules present on the virus or target cells but was completely blocked by polyanions such as heparin, dextran sulfate, and pentosan sulfate. Treatment of HeLa-CD4 cells with heparinases completely eliminated HIV attachment and infection, strongly implicating cell surface heparans in the attachment process. CD4 dependence for HIV-1 attachment to target cells is thus highly cell line specific and may be replaced by other ligand-receptor interactions.     

CDADC1在人视网膜色素上皮细胞中功能研究

目的：研究细胞增殖相关基因CDADC1在人视网膜色素上皮细胞ARPE19中的表达及对ARPE19细胞增殖的影响。方法：采用基因重组技术构建荧光表达载体pEGFP－C1－CDADC1和真核表达载体pcDNA3．1-myc-CDADC1,用脂质体转染法转染ARPE19细胞,观察GFP—CDADC1融合蛋白在ARPE19细胞的表达定位,流式细胞仪测定CDADC1转染后对ARPE19细胞生长周期、凋亡的影响。结果：FP—CDADC1融合蛋白亚细胞定位显示,CDADC1低表达于ARPE19细胞胞浆,高表达于细胞核;pcD—NA3．1-myc-CDADC1瞬时转染ARPE19细胞显示24小时细胞无明显改变,48小时后重组质粒转染组S期细胞占细胞总数的19．37％,pcDNA3．1-myc空载质粒转染组S期细胞占10．87％,而空白对照组S期细胞占3．33％,重组质粒转染组与两对照组之间的差异有统计学意义（P〈0．01）。结论：CDADC1在增生性玻璃体视网膜病变（PVR）发生和发展过程中可促进DNA的合成,引起细胞增生。