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At least 253 records · Page 14Linked to original sources

PubMed1976

Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA.

The translation of encephalomyocarditis virion RNA in extracts from interferon-treated L-cells is inhibited by the addition of double-stranded RNA (dsRNA) at 400 ng/ml. A similar inhibition in response to dsRNA is seen in control cell extracts supplemented with small amounts of a postribosomal supernatant fraction from interferon-treated cells (interferon cell sap): Neither interferon cell sap nor dsRNA alone is inhibitory in control systems. The inhibition is much reduced if translation is carried out at low ATP concentrations.Conversely, the inhibitory capacity of the interferon cell sap is increased 100-fold if it is preincubated with dsRNA and ATP prior to its addition to the protein-synthesizing system. After this preincubation all detectable dsRNA can be removed without any diminution of the inhibitory activity of the cell sap. These results are compatible with a two-step model for the inhibition in which a pre-inhibitor is activated by dsRNA, the activated inhibitor then interacting with the protein synthesis system to inhibit translation.

Adenosine Triphosphate↗

PubMed2004

Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract.

We describe a cell-free chromatin assembly system derived from the yeast Saccharomyces cerevisiae, which efficiently packages DNA into minichromosomes in a reaction dependent on exogenous core histones and an ATP-regenerating system. Both supercoiled and relaxed plasmid DNA serve as templates for nucleosomal loading in a gradual process that takes at least 6 h for completion at 30 degrees C. Micrococcal nuclease digestion of the assembled minichromosomes displays an extended nucleosomal ladder with a repeat length of 165 bp. The purified minichromosomes contain the four core histones in stoichiometric proportion and exhibit phased nucleosomes over the mouse mammary tumour virus (MMTV) promoter. The progesterone receptor and NF1 synergize on these minichromosomes resulting in efficient cell-free transcription. The ease of manipulation and the potential use of yeast strains carrying mutations in the chromatin handling machinery make this system suitable for detailed mechanistic studies.

Animals↗

PubMed1992

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

Animals↗

PubMed2004

Monitoring base excision repair proteins on damaged DNA using human cell extracts.

BER (base excision repair) is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). We have developed an assay, using formaldehyde cross-linking during repair in human cell extracts, to observe BER proteins involved in the repair of damaged DNA. This approach allows visualization of repair proteins on damaged DNA during BER in human cell extracts and provides a detailed view of the molecular events leading to repair.

DNA Damage↗

PubMed2005

Mammalian transcription activation domains of VP16, AP2 and CTF activate transcription in a whole cell extract from Schizosaccharomyces pombe through the SRB/mediator.

The acidic-rich activation domain of VP16 and the proline-rich activation domains of human AP2 and human CTF are able to activate transcription in a whole cell extract from Schizosaccharomyces pombe, whereas the glutamine-rich domains of Sp1 and Oct2 are unable to activate transcription in this system. Immunodepletion experiments of the whole cell extracts using specific antibodies against pombe TAF110, pombe TAF 72, pombe TBP and Srb4 shows that the activation of transcription by VP16, AP2 and CTF is through the mediator, since depletion of Srb4 inhibits activated transcription but does not inhibit basal transcription. Immunodepletion of TBP causes inhibition of both activated and basal transcription. On the other hand, immunodepletion of TAFs does not have an effect on either activated or basal transcription. Purified RNA polymerase holoenzyme is able to rescue the transcriptional activation activity of the anti-Srb4 immunodepleted extract. Moreover, we demonstrate that the mediator is needed for basal transcription of a TATA-less promoter.

Animals↗

PubMed1976

Influence of leukaemic cells on the colony formation of human bone marrow cells in vitro II. Suppressive effects of leukaemic cell extracts.

The influence of leukaemic cells on the colony formation of human bone marrow cells was studied in vitro as an extension of our previous work (Chiyoda et al., 1975). An extract of leukaemic bone marrow cells significantly suppressed colony forming ability of the normal bone marrow cells, whereas an extract of normal bone marrow cells did not suppress it except in two cases. The suppressive effect of normal bone marrow cells, however, was obviously less intense than that of leukaemic cells. This suppressive effect was dose dependent and was fairly stable to heat treatment. These results suggest that leukaemic bone marrow cells contain factor(s) which suppress normal colony formation.

Bone Marrow↗

PubMed1996

Measurement of Calcineurin Phosphatase Activity in Cell Extracts

The calmodulin-dependent phosphatase calcineurin is an important molecular target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. Complexes of CsA and FK506 with their immunophilin ligands interact with calcineurin in vitro, resulting in the inhibition of its serine/threonine phosphatase activity toward polypeptide substrates. In order to demonstrate that CsA and FK506 inhibit calcineurin in vivo, we developed an assay to measure calcineurin phosphatase activity in crude cell extracts. We have previously reported that incubation of intact cells with nanomolar concentrations of either CsA or FK506 results in potent inhibition of calcineurin activity in a variety of cell lines. Here we discuss in detail the methodology and applications of the cell extract calcineurin assay.

Journal Article↗

PubMed1993

Electron microscopy of DNA excision repair patches produced by human cell extracts.

To characterize the process by which the mammalian nucleotide excision repair complex interacts with DNA to recognize and repair lesions, we have investigated the size and distribution of repair patches induced by human cell extracts in ultraviolet light-irradiated plasmid DNA. Repair synthesis was carried out in a buffer substituting biotinylated dUTP for dTTP, to allow repair patches to be detected by electron microscopy after streptavidin/colloidal gold labelling. Individual repair events on circular plasmids that had undergone repair synthesis in cell extracts were scored as gold particles bound specifically to irradiated molecules. Samples of over 2000 irradiated and unirradiated plasmids were counted. Repair synthesis at ultraviolet light photoproducts typically replaced about 30 nucleotides, since 69% of patches contained only one particle of 10 nm gold and 24% of patches contained two gold particles (each covering approx. 29 nucleotides). In addition, the ordering of repair events among damaged plasmids closely fitted a Poisson distribution, indicating that repair of lesions is achieved via a non-processive, random diffusion mechanism. This suggests that the repair complex is not intrinsically processive.

Biotin↗

PubMed1990

Inhibition of platelet aggregation by a whole cell extract from strains of group B streptococcus.

A whole cell extract (HCl-Ext) from strains of group B streptococci (GBS) possessing fibrinogen binding activity prevented the platelet aggregation induced with adenosine 5'-diphosphate (ADP), collagen and thrombin, while aggregation by epinephrine and ristocetin was slightly inhibited and arachidonic acid-induced platelet aggregation was not affected whatsoever. When the HCl-Ext was added after commencement of the aggregation, deaggregation was observed in cases induced by ADP, collagen, and thrombin. By precoating the washed platelets with HCl-Ext, both of ADP- and collagen-induced platelet aggregation were suppressed. The active factor in the HCl-Ext seemed to be undialyzable, trypsin-susceptible, and proteinaceous substance, unlike GBS polysaccharide type antigen.

Fibrinogen↗

PubMed2003

Scintillation proximity assay of inositol phosphates in cell extracts: high-throughput measurement of G-protein-coupled receptor activation.

The phosphatidylinositol turnover assay is used widely to measure activation, and inhibition, of G(q)-linked G-protein-coupled receptors. Cells expressing the receptor of interest are labeled by feeding with tritiated myo-inositol. The label is incorporated into cellular phosphatidylinositol 4,5-bisphosphate, which, upon agonist binding to the receptor, is hydrolyzed by phospholipase C to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol. In the presence of Li(+), dephosphorylation of IP(3) to inositol is blocked, and the mass of soluble inositol phosphates is a quantitative readout of receptor activation. Current protocols for this assay all involve an anion-exchange chromatography step to separate radiolabeled inositol phosphates from radiolabeled inositol, making the assay cumbersome and difficult to automate. We now describe a scintillation proximity assay to measure soluble inositol phosphate mass in cell extracts, thus obviating the need for the standard chromatography step. The method uses positively charged yttrium silicate beads that bind inositol phosphates, but not inositol. We have used this assay to measure activation of recombinant and endogenous muscarinic acetylcholine receptors and activation of recombinant neuropeptide FF2 receptor coupled to IP(3) production by coexpression of a chimeric G protein. Further, we demonstrate the use and functional validity of this assay in a semiautomated, 384-well format, by characterizing the muscarinic receptor antagonists pirenzepine and atropine.

Animals↗

PubMed1996

Cytokeratin labeling of breast cancer cells extracted from paraffin-embedded tissue for bivariate flow cytometric analysis.

Flow cytometric analyses of DNA content and proliferative fractions have been found to be important prognostic indicators in a variety of human tumors. However, variability in reported results and interlaboratory differences in single-parameter DNA measurements have impeded the broader use of this methodology. Multiparameter DNA analysis, especially that which allows the ploidy and cell cycle measurements to be targeted specifically to tumor cells, may improve the quality and reliability of these measurements. Cytokeratin labeling simultaneously with DNA allows the identification of the malignant epithelial cells within tumor samples that have been microdissected for tumor enrichment and, thus, can increase the accuracy of tumor ploidy and cell cycle measurements. There are a limited number of reports of cytokeratin labeling of paraffin-extracted cells, and results with standard preparation procedures can be highly variable. We have developed an improved technique for cytokeratin labeling of paraffin-extracted cells that is based on predigestion of tissue with collagenase prior to brief pepsin digestion. This two-step enzymatic digestion produces a superior cytokeratin vs. DNA bivariate analysis, with increased intensity and greater uniformity of cytokeratin labeling. This method should increase the accuracy of ploidy and proliferation measurements from paraffin-embedded tissue both for retrospective studies and in clinical settings in which only fixed tissue is available.

Aneuploidy↗

PubMed1986

Use of sodium deoxycholate to extract cell wall components of virulent Mycobacterium bovis.

Mycobacterium bovis ATCC No. 19210 was grown on Middlebrook 7H-10 medium with pyruvate. Cells were harvested, and extracts were prepared, using 2% sodium deoxycholate (DOC) and 0.003M EDTA (in 0.1M Tris-HCl, 0.15M NaCl with 0.02% sodium azide [pH 8.4]). Phenylmethylsulfonyl fluoride was added, and the cells were extracted for 48 hours at 4 C. Cells were removed by centrifugation at 10,000 X g for 30 minutes. The supernatant was filter sterilized and separated into 2 fractions (peak A and peak B) by size-exclusion chromatography. The nonfractionated DOC extract and DOC peak A elicited delayed-type hypersensitivity responses at each of the protein concentrations tested (0.5, 1.5, and 4.5 micrograms) in M bovis-sensitized guinea pigs; responses were not detected, using DOC peak B. Significant differences were detected for each of the antigens when enzyme-linked immunosorbent assay values were compared, using sera from cattle before and 10 months after they were exposed to M bovis (P less than 0.01).

Animals↗

PubMed1992

Repair synthesis by human cell extracts in cisplatin-damaged DNA is preferentially determined by minor adducts.

During reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, a number of adducts are formed which may be discriminated by the excision-repair system. An in vitro excision-repair assay with human cell-free extracts has been used to assess the relative repair extent of monofunctional adducts, intrastrand and interstrand cross-links of cis-DDP on plasmid DNA. Preferential removal of cis-DDP 1,2-intrastrand diadducts occurred in the presence of cyanide ions. In conditions where cyanide treatment removed 85% of total platinum adducts while approximately 70% of interstrand cross-links remained in plasmid DNA, no significant variation in repair synthesis by human cell extracts was observed. Then, we constructed three types of plasmid DNA substrates containing mainly either monoadducts, 1,2-intrastrand cross-links or interstrand cross-links lesions. The three plasmid species were modified in order to obtain the same extent of total platinum DNA adducts per plasmid. No DNA repair synthesis was detected with monofunctional adducts during incubation with human whole cell extracts. However, a two-fold increase in repair synthesis was found when the proportion of interstrand cross-links in plasmid DNA was increased by 2-3 fold. These findings suggest that (i) cis-DDP 1,2-intrastrand diadducts are poorly repaired by human cell extracts in vitro, (ii) among other minor lesions potentially cyanide-resistant, cis-DDP interstrand cross-links represent a major lesion contributing to the repair synthesis signal in the in vitro assay. These results could account for the drug efficiency in vivo.

Cell Extracts↗

PubMed2006

Live cell extraction and HPLC-MS analysis for predicting bioactive components of traditional Chinese medicines.

A novel strategy for predicting bioactive components in traditional Chinese medicines (TCM) using live cell extraction and high performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) analysis was proposed. The hypothesis is that when cells are incubated together with the extract of TCM, the potential bioactive components in the TCM should selectively combine with the cells, and the relative concentrations of the cell-combining components in the suspension medium should decrease, while the cell-combining components would be detectable in the extract of denatured cells. The identities of the cell-combining components could be determined by HPLC-DAD-MS analysis. Using the proposed approach, the potential bioactive components of Danggui Buxue decoction, a commonly used TCM for anaemia, and its compositions, Radix Angelica Sinensis and Radix Astragli for endothelial cells, were investigated. Six compounds in the extract of Danggui Buxue decoction were detected as the components selectively combined with endothelial cells, among them two were contributed by Radix Angelica Sinensis, and four by Radix Astragli. The identities of four of the six potential bioactive compounds were elucidated as ononoside, calycosin, 3-butylphthalide and ligustilide by HPLC-DAD-MS analysis. The results indicate that the proposed approach may be applied to predict the bioactive candidates in TCM.

4-Butyrolactone↗

PubMed2002

Real-time determination of telomerase activity in cell extracts using an optical biosensor.

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3'-end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS)-modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signal-to-noise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.

Base Sequence↗

PubMed1994

Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts.

We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirectional replication of double-stranded DNA in a human cell extract. Plasmid vectors were constructed containing the simian virus 40 origin of replication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene. The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80%. Replication product analyses reveal unique termination sites with each damaged vector, suggesting that when the replication fork encounters an AAF adduct, it often stops before incorporation opposite the adduct. We also observed a higher proportion of products representing replication of the undamaged strand compared to the damaged strand. This suggests that the undamaged strand is replicated more readily, either by uncoupling the first fork to encounter the lesion or by replication using the fork arriving from the other direction. Also included among replication products are covalently closed monomer-length molecules resistant to cleavage at the AAF-modified Nar I site. This resistance is characteristic of substrates containing the AAF adduct, suggesting that translesion bypass had occurred. Transformation of Escherichia coli cells with the replicated damaged DNA yielded lacZ alpha revertant frequencies significantly above values obtained with undamaged DNA or with damaged DNA not replicated in vitro. This increase was only seen with the substrate modified at the third guanine position. Analysis of mutant DNA demonstrated the loss of a GC dinucleotide at the Nar I sequence. Generation of this position-dependent AAF-induced frameshift error in a human replication system is consistent with previous observations in E. coli suggesting that, after incorporation of dCMP opposite modified guanine in the third position, realignment of the template-primer occurs to form an intermediate with two unpaired nucleotides in the template strand.

2-Acetylaminofluorene↗

PubMed2002

In vitro (CTG)*(CAG) expansions and deletions by human cell extracts.

The mechanism of disease-associated (CTG)*(CAG) expansion may involve DNA replication slippage, replication direction, Okazaki fragment processing, recombination, or repair. A length-dependent bias for expansions is observed in humans affected by a trinucleotide repeat-associated disease. We developed an assay to test the effect of replication direction on (CTG)*(CAG) instabilities incurred during in vitro (SV40) DNA replication mediated by human cell extracts. This system recapitulates the bias for expansions observed in humans. Replication by HeLa cell extracts generated expansions and deletions that depended upon repeat tract length and the direction of replication. Templates with 79 repeats yielded predominantly expansions (CAG as lagging strand template) or predominantly deletions (CTG as lagging strand template). Templates containing 17 repeats were stable. Thus, replication direction determined the type of mutation. These results provide new insights into the orientation of replication effect upon repeat stability. This system will be useful in determining the contribution of specific human proteins to (CTG)*(CAG) expansions.

Blotting, Southern↗

PubMed1990

Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II.

We report the development of a new method for producing mitotic extracts from tissue culture cells. These extracts reproducibly promote the condensation of chromatin in vitro when incubated with purified interphase nuclei. This condensation reaction is not species specific, since nuclei from chicken, human, and hamster cell lines all undergo chromatin condensation upon incubation with the extract. We have used this extract to investigate the role of DNA topoisomerase II (topo II) in the chromosome condensation process. Chromatin condensation does not require the presence of soluble topo II in the mitotic extract. However, the extent of formation of discrete chromosome-like structures correlates with the level of endogenous topo II present in the interphase nuclei. Our results further suggest that chromatin condensation in this extract may involve two processes: chromatin compaction and resolution into discrete chromosomes.

Animals↗