SEARCH · PubMed Health

Results for “Cell Extracts”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources

PubMed2001

Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts.

The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 lacZ alpha DNA-based reversion assay. Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZ alpha gene. Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E. coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred. Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides. The misincorporation frequency of BER DNA synthesis at the target site was 5.2 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to G > T to A >> T to C. Uracil-initiated BER DNA synthesis in extracts of E. coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined. Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 x 10(-4). A reduced, but detectable level of BER was observed in extracts of E. coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold.

Adenocarcinoma↗

PubMed1988

Several aspects of total genomic DNA transcription in a HeLa cell extract.

Several aspects of total genomic DNA transcription in a HeLa cell extract were described. (1) By using in vitro transcripts from total genomic DNA as probes, we elucidated several structures of short interspersed elements which are transcribed by RNA polymerase III (designated as Pol III/SINEs. (2) To know whether the repetitive sequence we cloned and sequenced comprises a major or minor family, we developed in vitro run-off transcription assay of total genomic DNA. (3) Unlike in vitro transcription of total genomic DNA from vertebrates, major in vitro transcripts from macronuclear total DNA of Tetrahymena were found to be tRNA themselves.

Animals↗

PubMed1993

Partial purification and characterization of a polyketide biosynthetic enzyme, 6-hydroxymellein synthase, in elicitor-treated carrot cell extracts.

6-Hydroxymellein synthase, an induced polyketide biosynthetic enzyme in carrot cell extracts, was purified about 240-fold and its properties were compared with those of fatty acid synthetases. Synthetic activity of 6-hydroxymellein was inhibited in the presence of sulfhydryl reagents; however, cerulenin, a well-known inhibitor of fatty acid synthetases, showed no inhibitory activity to the enzyme. Biosynthesis of 6-hydroxymellein includes an NADPH-dependent ketoreduction, and, in this reaction, the 4-pro-S-hydrogen of NADPH was specifically transferred to the compound. On the basis of stereochemical analyses of the biosynthetic process, it was concluded that the product of the ketoreduction is an optically active alcohol of R configuration. These stereo-specificities of the reduction process are identical to those of beta-ketoacyl reductase in fatty acid biosynthesis which are considered to be conserved in all organisms. The synthetic rate of 6-hydroxymellein was markedly reduced when the assay was carried out with deuterium-labeled NADPH. The observed isotope effect on the catalytic rate (kH/kD) was 5.20, suggesting that this ketoreduction is one of the rate-limiting processes in 6-hydroxymellein synthesis. More than 85% of the synthetic activity was found in the soluble fraction of carrot cells, and, unlike in fatty acid synthetases in higher plants, organelle-localizing activity was not observed.

Acyltransferases↗

PubMed1984

The duck alpha A globin but not the yeast actin gene is transcribed by a HeLa cell extract.

We have investigated the transcription in a HeLa whole-cell extract of two evolutionary widely separated structural genes coding for duck alpha A globin and yeast actin. Transcription of isolated DNA fragments of the duck alpha A globin gene increases linearly up to relatively high concentrations of DNA. Size analyses and S1 mapping of the transcripts synthesized in vitro on either linear DNA fragments or supercoiled templates reveal that the alpha A globin RNA is initiated at the in vivo cap site and remains unspliced. The same assay conditions were used to transcribe the yeast actin gene. In contrast to the duck gene, size analyses and S1 mapping of the RNA products synthesized on both linear DNA fragments and the supercoiled template containing the actin gene show that the transcripts found in vitro do not stem from the in vivo cap site. The promoter of the yeast actin gene is not recognized in this system in vitro.

Actins↗

PubMed1998

Inhibition of human peripheral blood mononuclear cell proliferation by Streptococcus pyogenes cell extract is associated with arginine deiminase activity.

Streptococcus pyogenes (group A Streptococcus) cell extracts (CE) have a remarkably powerful and dose-dependent inhibitory effect on antigen, superantigen, or mitogen-stimulated human peripheral blood mononuclear cell (PBMC) proliferation in vitro. Purification of the inhibitory component present in S. pyogenes type M5 (Manfredo strain) CE by anion-exchange chromatography followed by gel filtration chromatography showed that the inhibitor had an approximate native molecular mass of 100 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitory fractions followed by silver staining gave a single band with an approximate molecular mass of 47 kDa, indicating that the inhibitor is composed of two identical subunits. NH2-terminal sequencing of the protein revealed that it was identical to the previously characterized streptococcal acid glycoprotein (SAGP); this protein possesses between 31.5 and 39.0% amino acid identity with arginine deiminase (AD) from Mycoplasma hominis, Mycoplasma arginini, Pseudomonas putida, and Pseudomonas aeruginosa. AD enzyme activity was present in unfractionated CE prepared from a range of streptococcal strains, and partially purified inhibitory fractions of Manfredo CE also had high levels of activity. The inhibitory effect of Manfredo CE was overcome by the addition of L-arginine to proliferation assays in which human PBMC were stimulated with phytohemagglutinin. We conclude that SAGP, or its homolog, possesses AD activity and that the potent inhibition of proliferation of human T cells by streptococcal CE is due to activity of this enzyme.

Amino Acid Sequence↗

PubMedDate not supplied

Uncovering the unfoldome: enriching cell extracts for unstructured proteins by acid treatment.

A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment where also shown experimentally to be totally disordered. A near 100 000-fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble E. coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver-stained 2-D SDS-PAGE gels loaded with 10 microg of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than 3-fold higher yield. A substantial yield of unprecipitated protein was obtained after 3% TCA treatment, suggesting that the common use of TCA precipitation prior to 2-D gel analysis may result in loss of unstructured protein due to their failure to precipitate. Our preliminary analysis suggests that treating total protein extracts with 3-5% PCA and determining the identities of soluble proteins could be the starting point for uncovering unfoldomes (the complement of unstructured proteins in a given proteome). The 100 000-fold reduction in yield and concomitant reduction in number of proteins achieved by 5% PCA treatment produced a fraction suitable for analysis in its entirety using standard proteomic techniques. In this way, large numbers of totally unstructured proteins could be identified with minimal effort.

Bacterial Proteins↗

PubMed1987

RNA end-labeling and RNA ligase activities can produce a circular rRNA in whole cell extracts from trypanosomes.

We have found two enzymatic activities in whole cell extracts from Trypanosoma brucei; an end-labeling reaction involving a single uridine at the 3' end of ribosomal RNAs (rRNAs) and an RNA ligase activity joining 5' monophosphates to 3' hydroxyl groups. The RNA ligase acts upon one of the small rRNAs (180 nucleotides) from the trypanosome ribosomal repeat unit, forming a circular RNA. The specific circularization of this small rRNA is probably dependent on the secondary structure of the molecule and is not detectable in vivo.

Animals↗

PubMedDate not supplied

[Reactivation of Escherichia coli inactivated by ultraviolet light by cell extracts of propionic acid bacteria].

For the first time reactivation of cell extract of three strains of Propionibacterium shermanii in UV inactivated not filament-forming strain Escherichia colli AB 1157 is shown. Reactivation was demonstrated in preincubated and postincubated test-culture and increased as survival of E. coli decreased in a range 1.8-0.006%. The factor (factors) of defense is dialysable, thermolabile and is present as in a fraction of nucleoproteins and nucleic acids so in a fraction of soluble proteins. The extracts were inactivated by incubation with proteinase K and trypsin, partly decreased activity by incubation with alpha-amylase and selected nuclease but not with lipase. Polypeptide nature of reactivating factor is supposed.

Carbon↗

PubMed2005

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.

Base Sequence↗

PubMed2006

Screening and analysis of bioactive compounds in traditional Chinese medicines using cell extract and gas chromatography-mass spectrometry.

As the cost of drug development is always many times more than that of drug discovery, predictive methods aiding in the screening of bioavailable drug candidates are of profound significance. In this paper, a novel method for screening bioactive compounds from traditional Chinese medicines (TCMs) was developed by using living cell extract and gas chromatography (GC)-mass spectrometer (MS). The method was validated by using elemene emulsion injection (EEI), a typical TCM with known active compound, to interact with murine ascites hepatocarcinoma cell strain with high metastatic potential (HCa-F). Finally, the method was applied to screen the bioactive compounds from multi-component zedoary turmeric oil and glucose injection (ZTOGI). After HCa-F cells was incubated in ZTOGI, ethyl acetate (EtOAc) was used to extract the compounds in the cells for GC-MS analysis. Fourteen compounds were detected in the desorption eluate of HCa-F cell extract of ZTOGI, and further identified by MS. Curzerene and beta-elemene were found to be two major bioactive compounds in ZTOGI. These results show that the method developed may be applied to quickly screen the potential bioactive components in TCMs interacting with the target cells.

Animals↗

PubMed2006

Method to determine 4-oxo-retinoic acids, retinoic acids and retinol in serum and cell extracts by liquid chromatography/diode-array detection atmospheric pressure chemical ionisation tandem mass spectrometry.

Retinoic acid isomers, 4-oxo-retinoic acid isomers and retinol are present in the serum of mammals. In this study a high-performance liquid chromatography (HPLC) separation, sample preparation and tandem mass spectrometry (MS/MS) method was established for quick and easy sample preparation and sensitive determination of retinoids such as all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid and retinol in serum and cell extracts. Serum samples were simply treated with three times the volume of isopropanol, dried under vacuum, taken up in the HPLC solvent and immediately put into the autosampler for an automated single-run HPLC analysis. With this MS/MS method we were able to detect 7 pg and quantify 20 pg of all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid and retinol directly on-column and were able to determine a concentration as low as 0.2 ng/mL in ethanolic standards and in biological samples. This method allows ultra-sensitive detection, excellent selectivity and a very simple sample preparation to determine retinoic acids, 4-oxo-retinoic acids and retinol in serum and cell extracts for the study of endogenous retinoids.

Atmospheric Pressure↗

PubMed2005

Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins.

Clustered DNA lesions, possibly induced by ionizing radiation, constitute a trial for repair processes. Indeed, recent studies suggest that repair of such lesions may be compromised, potentially leading to the formation of lethal double-strand breaks (DSBs). A complex multiply damaged site (MDS) composed of 8-oxoguanine and 8-oxoadenine on one strand, 5-hydroxyuracil, 5-formyluracil and a 1 nt gap on the other strand, within 17 bp was built and used to challenge several steps of base excision repair (BER) pathway with human whole-cell extracts and purified repair enzymes as well. We show a hierarchy in the processing of lesions within the MDS, in particular at the base excision step. In the present configuration, efficient excision of 5-hydroxyuracil and low cleavage at 8-oxoguanine prevent DSB formation and generate a short single-stranded region carrying the 8-oxoguanine. On the other hand, rejoining of the 1 nt gap occurs by the short-patch BER pathway, but is slightly retarded by the presence of the oxidized bases. Taken together, our results suggest a hierarchy in the processing of the lesions within the MDS, which prevents the formation of DSB, but would dramatically enhance mutagenesis. They also indicate that the mutagenic (or lethal) consequences of a complex MDS will largely depend on the first event in the processing of the MDS.

Bacteria↗

PubMed1988

Processing at immunoglobulin polyadenylation sites in lymphoid cell extracts.

We have developed an in vitro system for polyadenylation of RNA substrates in cell-free nuclear extracts prepared from murine cells of lymphoid origin. RNA substrates containing the adenovirus L3, murine immunoglobulin (IgM) secreted and membrane polyadenylation sites were accurately polyadenylated in these extracts. Kinetic analysis showed that the rate of polyadenylation in vitro responds proportionally to the substrate concentration. Quantitation of the initial rate of polyadenylation at the three sites permitted comparison of the activities of extracts prepared from HeLa cells, B cells (Wehi 231) and plasmacytoma cells (P9.37.11). From this analysis, we concluded that in all three extracts the polyadenylation activity at the L3 site was higher than that of either of the IgM sites. In contrast to the preferential utilization of the secreted site in vivo in plasmacytomas, this site was not selectively processed in plasmacytoma as compared to B cell extracts. The efficiency of polyadenylation at both IgM sites in the plasmacytoma extract was significantly lower than that in the B cell extract. The common low activity at the IgM sites in the plasmacytoma cell extract suggests that the rate-limiting step for polyadenylation at these two sites differs from that at the L3 site.

Animals↗

PubMed2006

Toxic effects of Microcystis cell extracts on the reproductive system of male mice.

In this paper, the reproductive toxicity of male mice treated with Microcystis aeruginosa cell extracts containing microcystins was examined. In contrast to the control group, male mice exposed intraperitoneally to 3.33 or 6.67 microg microcystins/kg body weight for 14 days had decreased mean body weight, and the mean absolute weight of the testes and epididymides was decreased. However, the mean relative weight of the testes increased compared to the controls. In addition, histological examination of microcystin-treated mice indicated that the testes were damaged and the space between the seminiferous tubules was more pronounced compared to control mice. The quality of mature sperm in the seminiferous tubules was also decreased in treated mice compared with the control group. Further studies showed that motility and viability of the sperm from microcystin-treated mice were reduced, but no significant difference was found in the concentration and abnormality of the sperm from treated mice compared to the control. This study indicated that microcystins had numerous toxic effects on the reproductive system of male mice.

Animals↗

PubMed2001

Enzymatic determination of homocysteine in cell extracts.

Determination of homocysteine levels in cells and serum is important because high homocysteine is a risk factor for cardiovascular disease. The currently used methods for homocysteine analysis either are time consuming or rely on the use of expensive equipment. Described in this study is an enzymatic assay that determines levels of homocysteine in multiple samples in less than 30 min at levels from 5 to 50 pmol using only a spectrophotometer. The reproducibility of the assay is consistent with the other methods currently used. A second assay, that is about 5-fold more sensitive, follows the enzymatic catalyzed solvent exchange of protons on glycine, which requires a scintillation counter. Both the spectrophotometric and the radiometric methods are based on the conversion of 5-methyltetrahydrofolate to tetrahydrofolate by methionine synthase. The tetrahydrofolate is formed in stoichiometric amounts to the homocysteine in the sample. In the spectrophotometric method the tetrahydrofolate is used at catalytic levels by three enzymes to form a metabolic cycle that generates NADPH from NADP(+). In the radiometric assay tetrahydrofolate is required for the enzymatic exchange of the pro 2S proton of glycine with solvent. L-Cysteine, at levels more than 30-fold higher than the upper level of homocysteine used in these assays, does not give any measurable response.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1993

Repair by human cell extracts of single (6-4) and cyclobutane thymine-thymine photoproducts in DNA.

One cis-syn cyclobutane thymine dimer or one (6-4) thymine-thymine photoproduct was built into an identical sequence of a closed-circular M13 duplex DNA, and nucleotide excision repair synthesis carried out by human cell extracts in the area containing each lesion was determined. Extracts from normal cells repaired the (6-4) photoproduct with a patch size of approximately 20-30 nucleotides, but repair was at least 10-fold lower at the cyclobutane dimer. The (6-4) lesion was repaired with comparable efficiency to a single acetylamino-fluorene-guanine adduct in a similar location. Extract from nucleotide excision repair-deficient xeroderma pigmentosum group A cells could not remove any of these adducts but could complete repair of the lesions after incision with Escherichia coli UvrABC proteins. This direct comparison of repair of two UV photoproducts, in an in vitro system where chromatin assembly and transcription are absent, suggests that the more rapid repair of the (6-4) lesion observed in the mammalian cell genome overall is due in part to a significant difference in the ability of the repair complex to locate and incise these lesions in DNA.

Bacteriophage M13↗

PubMed1997

Replication of M13 single-stranded viral DNA bearing single site-specific adducts by escherichia coli cell extracts: differential efficiency of translesion DNA synthesis for SOS-dependent and SOS-independent lesions.

In order to characterize mutagenic translesion DNA synthesis in UVM-induced Escherichia coli, we have developed a high-resolution DNA replication system based on E. coli cell extracts and M13 genomic DNA templates bearing mutagenic lesions. The assay is based on the conversion of M13 viral single-stranded DNA (ssDNA) bearing a single site-specific DNA lesion to the double-stranded replicative form (RF) DNA, and permits one to quantitatively measure the efficiency of translesion synthesis. Our data indicate that DNA replication is most strongly inhibited by an abasic site, a classic SOS-dependent noninstructive lesion. In contrast, the efficiency of translesion synthesis across SOS-independent lesions such as O6-methylguanine and DNA uracil is around 90%, very close to the values obtained for control DNA templates. The efficiency of translesion synthesis across 3,N4-ethenocytosine and 1, N6-ethenoadenine is around 20%, a value that is similar to the in vivo efficiency deduced from the effect of the lesions on the survival of transfected M13 ssDNA. Neither DNA polymerase I nor polymerase II appears to be required for the observed translesion DNA synthesis because essentially similar results are obtained with extracts from polA- or polB-defective cells. The close parallels in the efficiency of translesion DNA synthesis in vitro and in vivo for the five site-specific lesions included in this study suggest that the assay may be suitable for modeling mutagenesis in an accessible in vitro environment.

Bacteriophage M13↗

PubMed1998

Some kinetic properties of lactate dehydrogenase activity in cell extracts from a mammalian (ovine) corneal epithelium.

The purpose of the following study was to examine the kinetic properties of lactate dehydrogenase (LDH) in cell extracts of corneal epithelium. The epithelium, from quality- and size-selected ewe corneas, was dispersed in 10 mM Trizma maleate with or without sucrose at pH 7.0 and cell fractions obtained by homogenisation and differential centrifugation for evaluation of LDH at 37 degrees C. Pyruvate-dependent oxidation of NADH was optimal at pH 6.5, while lactate-dependent reduction of NAD was optimal at pH 9.5; the former activity averaged 3 to 3.5 fold higher than the latter. NADPH and NADP were poor cofactors. The apparent Km values for pyruvate and lactate were 99 micromoles and 3.9 millimoles respectively. At pH 6. 5, substrate inhibition was observed with pyruvate in excess of 0.25 mm with 50% activity measured at 1.2 mm. Excess substrate effects were not seen with lactate at pH 9.5, but pyruvate inhibited lactate-dependent reduction of NAD with 50% activity measured at 1.1 mm. Differential centrifugation confirmed that the activity was predominantly localised in the soluble (post mitochondria-lysosome) fraction of the cells.

Animals↗