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A human embryonic kidney cell line 293 is widely used for adenovirus production and propagation. With this cell line, however, replication-competent virus (RCV) is frequently generated, especially during large-scale production and successive propagation because 293 cells contain not only E1 gene but also non-E1 adenovirus gene. Homologous recombination between non-E1 region of 293 genomic DNA and its homologous region in the recombinant adenoviral vector generate RCV. To overcome this problem, we developed a new packaging cell line, Hela-E1, which contains minimum E1 region and from which non-E1 adenoviral region that is homologous with recombinant adenovirus vector was excluded. No RCV was detected during adenovirus propagation in Hela-E1 compared to in 293. In addition, adenovirus-p53 produced in HeLa-E1 was able to overexpress p53 protein when introduced into an ovarian cancer cell line, SKOV3. These results may have a significant impact on the development of packaging cell lines for replication-deficient adenovirus production.  相似文献   

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The small‐DNA human adenovirus encodes one of the most versatile molecular hubs, the E1A protein. This protein is essential for productive viral infection in human cells and a vast amount of biologically relevant data are available on its interactions with host proteins. Up to now, however, no high‐resolution structural and dynamic information on E1A is available despite its important biological role. Among the different spliced variants of E1A, two are expressed at high level in the early stage of infection. These are 243 and 289 residues isoforms. Herein, we present their NMR characterization, showing that they are both highly disordered, but also demonstrate a certain heterogeneous behavior in terms of structural and dynamic properties. Furthermore, we present the characterization of the isolated domain of the longer variant, known as CR3. This study opens the way to understanding at the molecular level how E1A functions.  相似文献   

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Multifunctional gold nanoparticle-peptide complexes for nuclear targeting   总被引:7,自引:0,他引:7  
The ability of peptide-modified gold nanoparticles to target the nucleus of HepG2 cells was explored. Five peptide/nanoparticle complexes were investigated, particles modified with (1) the nuclear localization signal (NLS) from the SV 40 virus; (2) the adenovirus NLS; (3) the adenovirus receptor-mediated endocytosis (RME) peptide; (4) one long peptide containing the adenovirus RME and NLS; and (5) the adenovirus RME and NLS peptides attached to the nanoparticle as separate pieces. Gold nanoparticles were used because they are easy to identify using video-enhanced color differential interference contrast microscopy, and they are excellent scaffolds from which to build multifunctional nuclear targeting vectors. For example, particles modified solely with NLS peptides were not able to target the nucleus of HepG2 cells from outside the plasma membrane, because they either could not enter the cell or were trapped in endosomes. The combination of NLS/RME particles (4) and (5) did reach the nucleus; however, nuclear targeting was more efficient when the two signals were attached to nanoparticles as separate short pieces versus one long peptide. These studies highlight the challenges associated with nuclear targeting and the potential advantages of designing multifunctional nanostructured materials as tools for intracellular diagnostics and therapeutic delivery.  相似文献   

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Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia, cancer/leukemia susceptibility and cellular hypersensitivity to DNA crosslinking agents, such as cisplatin. To date, 12 FA gene products have been identified, which cooperate in a common DNA damage-activated signaling pathway regulating DNA repair (the FA pathway). Eight FA proteins form a nuclear complex harboring E3 ubiquitin ligase activity (the FA core complex) that, in response to DNA damage, mediates the monoubiquitylation of the FA protein FANCD2. Monoubiquitylated FANCD2 colocalizes in nuclear foci with proteins involved in DNA repair, including BRCA1, FANCD1/BRCA2, FANCN/PALB2 and RAD51. All these factors are required for cellular resistance to DNA crosslinking agents. The inactivation of the FA pathway has also been observed in a wide variety of human cancers and is implicated in the sensitivity of cancer cells to DNA crosslinking agents. Drugs that inhibit the FA pathway may be useful chemosensitizers in the treatment of cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).  相似文献   

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Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.  相似文献   

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Peng W  Yuan K  Zhou X  Hu M  Abs EL-Osta YG  Gasser RB 《Electrophoresis》2003,24(14):2308-2315
Using a single-strand conformation polymorphism-based approach, we investigated nucleotide variation in part of the first internal transcribed spacer (pITS-1) of nuclear ribosomal DNA within and among a large number of Ascaris individuals from humans and pigs from six endemic regions in China, and examined the frequency of the different genotypes of Ascaris in relation to host species and geographical origin. Five different SSCP genotypes (G1-G5) were recorded for human Ascaris (n = 486), of which three (genotypes G1-G3) were detected for pig Ascaris (n = 329). Of the five Ascaris genotypes detected, genotype G1 predominantly infected humans (approximately 63-74%) whereas genotype G3 infected mainly pigs (approximately 79-86%), indicating that each of these genotypes has a particular host affiliation. In contrast, the frequencies of the other three genotypes was substantially lower for each of the two host species. The findings also suggested that the rate of cross infection of Ascaris between humans and pigs is relatively low and that gene flow between the predominant genotypes is limited, consistent with previous proposals for endemic regions in other countries. While the nature and extent of nucleotide variation in the pITS-1 (and the proposal of host affiliated Ascaris populations) may relate to "introgression" or "lineage sorting and retention of ancentral polymorphism", other explanations are possible. Evidence of multiple pITS-1 sequence types in some Ascaris individuals representing particular genotypes (e.g., G2 and G5) may suggest hybridization between human- and pig-affiliated Ascaris. This aspect and the species status of Ascaris (from each host species) warrant future experimental testing, employing the pig/Ascaris model and the present electrophoretic approach.*  相似文献   

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To overcome limitations of conventional approaches for the identification of Eimeria species of chickens, we have established high resolution electrophoretic procedures using genetic markers in ribosomal DNA. The first and second internal transcribed spacer (ITS-1 and ITS-2) regions of ribosomal DNA were amplified by polymerase chain reaction (PCR) from genomic DNA samples representing five species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella), denatured and then subjected to denaturing polyacrylamide gel electrophoresis (D-PAGE) or single-strand conformation polymorphism (SSCP) analysis. Differences in D-PAGE profiles for both the ITS-1 and ITS-2 fragments (combined with an apparent lack of variation within individual species) enabled the unequivocal identification of the five species, and SSCP allowed the detection of population variation between some isolates representing E. acervulina, which remained undetected by D-PAGE. The establishment of these approaches has important implications for controlling the purity of laboratory lines of Eimeria, for diagnosis and for studying the epidemiology of coccidiosis.  相似文献   

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BACKGROUND: Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity. RESULTS: Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. CONCLUSIONS: We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.  相似文献   

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Abstract— UV-induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 103 particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV-induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.  相似文献   

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Detection of trace amounts of allergens is essential for correct labeling of food products by the food industry. PCR-based detection methods currently used for this purpose are targeting sequences of DNA present in the cell nucleus. In addition to nuclear DNA, a substantial amount of mitochondrial DNA (mtDNA) copies are present in the cytoplasm of eukaryotic cells. The nuclear DNA usually consists of a set of DNA molecules present in two copies per cell, whereas mitochondrial DNA is present in a few hundred copies per cell. Thus, an increase in sensitivity can be expected when mtDNA is used as the target. In this study, we present a reporter probe-based real-time PCR method amplifying the mitochondrial gene of the alpha chain of adenosine triphosphate synthetase from soy. Increase in sensitivity was examined by determining the minimal amount of soy DNA detectable by mtDNA and nuclear DNA (nDNA) amplification. Additionally, the LOD of soy in a food matrix was determined for mtDNA amplification and compared to the LOD determined by nDNA amplification. As food matrix, a model spice spiked with soy flour was used. Sensitivity of PCR-based soy detection can be increased by using mtDNA as the target.  相似文献   

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A new water soluble cationic imidazopyridine species, viz. (1E)-1-((pyridin-2-yl)methyleneamino)-3-(3-(pyridin-2-yl)imidazo[1,5-a]pyridin-2(3H)-yl)propan-2-ol (1), as a metal chelator is prepared as its PF(6) salt and characterized. Compound 1 shows fluorescence at 438 nm on excitation at 342 nm in Tris-HCl buffer giving a fluorescence quantum yield (φ) of 0.105 and a life-time of 5.4 ns. Compound 1, as an avid DNA minor groove binder, shows pUC19 DNA cleavage activity in UV-A light of 365 nm forming singlet oxygen species in a type-II pathway. The photonuclease potential of 1 gets enhanced in the presence of Fe(2+), Cu(2+) or Zn(2+). Compound 1 itself displays anticancer activity in HeLa, HepG2 and Jurkat cells with an enhancement on addition of the metal ions. Photodynamic effect of 1 at 365 nm also gets enhanced in the presence of Fe(2+) and Zn(2+). Fluorescence-based cell cycle analysis shows a significant dead cell population in the sub-G1 phase of the cell cycle suggesting apoptosis via ROS generation. A significant change in the nuclear morphology is observed from Hoechst 33258 and an acridine orange/ethidium bromide (AO/EB) dual nuclear staining suggesting apoptosis in cells when treated with 1 alone or in the presence of the metal ions. Apoptosis is found to be caspase-dependent. Fluorescence imaging to monitor the distribution of 1 in cells shows that 1 in the presence of metal ions accumulates predominantly in the cytoplasm. Enhanced uptake of 1 into the cells within 12 h is observed in the presence of Fe(2+) and Zn(2+).  相似文献   

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Abstract— The kinetics of enzymatic photoreactivation (PR) of u.v.-induced killing was compared among E. coli Bs-1, phage T1 in Bs-1 and phage T1 in irradiated Bs-1. The PR action spectrum showed no substantial difference between PR of Bs-1 and PR of T1 in Bs-1. The PR D37 (i.e. the PR dose required to reactivate all but 37 per cent of the reactivable lethal lesions) was found to decrease linearly with decreasing U.V. dose whether U.V. was given to produce pyrimidine dimers in Bs-1 DNA, which then compete with irradiated T1 DNA for PR enzyme, or to Bs-1 or T1 DNA to produce dimers serving as substrate for the PR enzyme. A generalized Michaelis-Menten formula was used to analyze the data and the following conclusions were drawn. (1) The number of PR enzyme molecules per cell available for PR of T1 DNA inside the Bs-1 host is only a quarter of the number available for PR of the Bs-1 host itself. (2) The Michaelis constant Km for reaction of host-DNA-damage and PR-enzyme becomes larger when the host damage acts as competitive inhibitor to PR of T1 DNA than when it is the substrate for PR enzyme. (3) PR enzyme retains almost all its initial catalytic efficiency even after about two-hundred rounds of catalytic functioning. Conclusions (1) and (2) suggest that PR enzyme is concentrated within the nuclear area surrounding the host DNA.  相似文献   

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Abstract— 4ells from patients with the sun sensitive cancer-prone disease, xeroderma pigmentosum (XP) have defective repair of UV damaged DNA with reduced excision of the major photoproduct, the cyclobutane type pyrimidine dimer. Other (non-dimer) photoproducts, have recently been implicated in UV mutagenesis. Utilizing an expression vector host cell reactivation assay, we studied UV damaged transfecting DNA that was treated by in vitro photoreactivation to reverse pyrimidine dimers while not altering other photoproducts. We found that the reduced expression of a UV damaged transfecting plasmid in XP complementation group A cells is only partially reversed by photoreactivation. E. coli photolyase treatment of pSV2catSVgpt exposed to 100 or 200 J m−2 of 254 nm radiation removed 99% of the T4 endonuclease V sensitive sites. Transfection of XP12BE(SV40) cells with photoreactivated pSV2catSVgpt showed residual inhibition corresponding to 25 to 37% of the lethal hits to the cat gene. This residual inhibition corresponds to the fraction of non-dimer photoproducts induced by UV. This result implies that XP12BE(SV40) cells do not repair most of the non-dimer photoproducts in DNA.  相似文献   

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A new method for the possible incorporation of nuclear wastes has been attempted here by using ceramic matrix of TiO2 as a host precursor for confinement. Hafnium is used as a simulant for actinide high-level waste. After incorporating 181Hf tracer into TiO2 matrix, the leaching property of the resulting matrix was studied in water, sodium chloride and humic acid solutions. The leaching was measured in each of the case by following the radioactivity of 181Hf. TiO2 matrix has also been exposed to γ-radiation in order to simulate the radiation field for nuclear waste. It has been investigated with a nuclear technique called time differential perturbed Angular Correlation (TDPAC) that the lattice structure of titania remains undisturbed even under a strong radiation field. The leaching of 181Hf has also been studied after irradiating the TiO2 matrix with γ-radiation and the leaching behavior was observed not to change from that before irradiation.  相似文献   

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A CZE method was validated and implemented for fast and accurate in‐process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus vector‐based vaccines. An analytical‐quality‐by‐design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing.  相似文献   

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