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 217 records · Page 12Linked to original sources

PubMed2001

A single (6-4) photoproduct inhibits plasmid DNA replication in xeroderma pigmentosum variant cell extracts.

The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in the human RAD30 gene, which encodes the lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV-irradiation. Using extracts of an XPV lymphoblast cell line (GM2449C) that has a truncating mutation in the RAD30 gene, we investigated the effect of a (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD), at a unique -TT- site on either the leading or lagging strand, on plasmid DNA replication. Compared to normal cell extracts, XPV cell extracts have a reduced capacity to carry out complete replication of DNA containing either a (6-4) photoproduct or a CPD on the leading strand, whereas there is little difference between the two cell extracts in replication of DNA containing a lesion on the lagging strand. Inhibition of replication in the presence of a (6-4) photoproduct is attributed to arrest of nascent DNA strand synthesis at the lesion site; in XPV cell extracts, the proportion of arrested products is increased compared to that of normal cell extracts. These results are consistent with a requirement for functional DNA polymerase eta in the replication of a double-stranded plasmid containing either a (6-4) photoproduct or a CPD, on the leading but not the lagging strand.

Base Sequence↗

PubMed2006

Molecular imprinted polymer with cloned bacterial protein template enriches authentic target in cell extract.

Here we describe a new method for preparing a protein-imprinted polymer with a cloned bacterial protein template, which recognizes/adsorbs authentic target protein present at a relatively low level in cell extract. In this work, cloned pig cyclophilin 18 (pCyP18) was used as a template. The template protein was selectively assembled with memory molecules from their library, which consists of numerous limited length polymer chains with randomly distributed recognition sites and immobilizing sites. These assemblies of protein and memory molecules were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, binding sites that were complementary to the target protein in size, shape and the position of recognition groups were exposed, and their confirmation was preserved by the cross-linked structure. The synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 300 times.

Bacterial Proteins↗

PubMed2005

Poly(ADP-ribose) polymerase-1 protects excessive DNA strand breaks from deterioration during repair in human cell extracts.

Base excision repair (BER), a major pathway for the removal of simple lesions in DNA, requires the co-ordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. We here address the role of poly(ADP-ribose) polymerase-1 (PARP-1) in BER. Using an in vitro cross-linking assay, we reveal that PARP-1 is always involved in repair of a uracil-containing oligonucleotide and that it binds to the damaged DNA during the early stages of repair. Inhibition of PARP-1 poly(ADP-ribosyl)ation by 3-aminobenzamide blocks dissociation of PARP-1 from damaged DNA and prevents further repair. We find that excessive poly(ADP-ribosyl)ation occurs when repair intermediates containing single-strand breaks are in excess of the repair capacity of the cell extract, suggesting that repeated binding of PARP-1 to the nicked DNA occurs. We also find increased sensitivity of repair intermediates to nuclease cleavage in PARP-deficient mouse fibroblasts and after depletion of PARP-1 from HeLa whole cell extracts. Our data support the model in which PARP-1 binding to DNA single-strand breaks or repair intermediates plays a protective role when repair is limited.

Animals↗

PubMed1991

Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins.

Adult↗

PubMed1984

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts.

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [gamma-32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

Animals↗

PubMed1996

Silencing of the Escherichia coli bgl promoter: effects of template supercoiling and cell extracts on promoter activity in vitro.

Regulation of the Escherichia coli bgl promoter involves the catabolite gene activator protein (CAP) and silencer elements that are located upstream and downstream of the promoter and its CAP binding site. The promoter is kept in a repressed state by the silencer elements and other normally active CAP-dependent or -independent promoters are repressed as well when flanked by these elements. To assess the mechanism of promoter repression, single round in vitro transcription was carried out with plasmids bearing either the wild-type bgl promoter or one of two derivatives that escape repression in vivo by different mechanisms: C234 by improving the CAP binding site of the promoter and delta1 by a deletion within the upstream silencer sequence. Repression of the bgl promoter in vitro was shown to depend on template topology and the presence of cellular factors. With negatively supercoiled templates, all three promoters are transcribed to similar extents by purified E. coli RNA polymerase and no CAP dependence is apparent; with relaxed templates, transcription is CAP dependent, but the levels of transcription of the three promoters are comparable. Addition of crude cell extract to the simple transcription system leads to repression of all three promoter alleles in the absence of CAP. Repression of the mutant alleles but not of the wild-type promoter is completely relieved in the presence of the CAP-cAMP complex. The topology of the DNA template is also important in the differential regulation of these promoters. In the case of C234, repression by cell extract is completely relieved by CAP-cAMP on relaxed or negatively supercoiled templates, while complete derepression of delta1 by CAP-cAMP occurs on negatively supercoiled templates only. Repression by cell extract requires the presence of the histone-like protein H-NS. However, H-NS alone does not appear to be sufficient for specific silencing of the wild-type promoter, since repression of all three promoter alleles caused by purified H-NS protein is completely relieved by the CAP-cAMP complex. These data suggest that template topology, H-NS and other cellular factors are involved in the formation of a specific nucleoprotein structure in the bgl promoter-silencer region; the formation of this nucleoprotein structure keeps an otherwise active promoter in an inactive state.

Amino Acid Sequence↗

PubMed1972

Activation of the viral genome in Simian Virus 40-transformed nonpermissive cells by permissive cell extracts.

Simian virus 40-transformed nonpermissive cells in which neither infectious viral DNA nor virions had previously been detected reproducibly yield low levels of infectious simian virus-40 DNA after treatment with extracts of normal permissive cells. Virions were obtained in one out of 37 experiments. Activation of the viral genome is detected only with normal permissive cell extracts and only in simian virus 40-transformed cell lines that are rescuable by heterokaryon formation or by treatment with chemicals. The activating factor(s) is insensitive to deoxyribonuclease and ribonuclease, but is sensitive to heat at 56 degrees and to proteolytic enzymes.

Animals↗

PubMed1986

Novel mutants of CHO cells resistant to adenosine analogs and containing biochemically altered form of adenosine kinase in cell extracts.

Stable mutants which are approximately five- and eightfold resistant to an inosine analog, formycin B (Fomr) have been selected in a single-step from Chinese hamster ovary cells at a frequency of approximately 10(-6). Cross-resistance studies with these mutants show that the Fomr mutants exhibit increased resistance to all adenosine analogs (N-and C-nucleosides) examined and, in accordance with their cross-resistance pattern, the mutants exhibited decreased cellular uptake and phosphorylation of formycin B and various adenosine analogs. In cell hybrids formed with sensitive cells, the drug-resistant phenotype of these mutants behaved recessively. However, unlike mutants resistant to adenosine analogs that have been obtained previously, which contain no measurable activity of adenosine kinase (AK) in cell extracts, the two Fomr mutants studied contained about 60 and 110% of the enzyme activity (compared to the parental cells) in their cell extracts. Biochemical studies with AK from the mutant cells show that in comparison to the wild-type enzyme, the mutant enzymes required much higher concentrations of the adenosine analog N7-(delta 2-isopentenyl) formycin A for similar inhibition of [3H]adenosine phosphorylation. These results indicate that AK from the Fomr mutants has lower affinity for phosphorylation of adenosine analogs in comparison to the enzyme from the parental cells. The genetic lesion in the Fomr mutants may thus be directly affecting the structural gene for AK.

Adenosine↗

PubMed2005

Isolation of uncultivated anaerobic thermophiles from compost by supplementing cell extract of Geobacillus toebii in enrichment culture medium.

Several researchers have reported that microorganisms can be cultivated only in the presence of other microorganisms. We suggest that a portion of uncultivated microorganisms might be cultivated in the presence of cellular components released from bacteria in their natural environments. In this study, the cell extract of Geobacillus toebii was used to enrich uncultivated thermophiles from compost. In the process of enrichment cultures, cell extract supplementation apparently changed the community composition. This change was monitored by PCR-DGGE targeting 16S rRNA gene. Five novel groups of microorganisms (similarity of 16S rRNA gene to the closest relative <96%) were specifically isolated from enrichment cultures by using cell extract-supplemented culture media. Their growth was found to be dependent on the addition of extract of G. toebii. Putting these findings together, we suggest that the extracts of bacteria could be one of the growth factors in the thermal ecosystem with a possibility of extending other ecological niches.

Bacillaceae↗

PubMed1992

Max and c-Myc/Max DNA-binding activities in cell extracts.

We have examined the interactions and DNA-binding activities of the c-Myc oncoprotein and its partner Max. In cell extracts virtually all c-Myc molecules are associated with Max in heterodimeric complexes. Moreover, DNA-binding studies with in vitro-translated protein and cell extracts show that both Max alone and c-Myc/Max bind the same DNA sequence. Conversely, c-Myc is unable to bind this sequence in the absence of Max. These findings suggest that c-Myc may function via obligate complex formation with Max.

Animals↗

PubMed1965

CHARACTERIZATION OF CONJUGATION FACTORS IN ESCHERICHIA COLI CELL WALLS. I. INHIBITION OF RECOMBINATION BY CELL WALLS AND CELL EXTRACTS.

Lancaster, John H. (University of Texas, Austin), E. P. Goldschmidt, and Orville Wyss. Characterization of conjugation factors in Escherichia coli cell walls. I. Inhibition of recombination by cell walls and cell extracts. J. Bacteriol. 89:1478-1481. 1965.-An assay procedure was devised to determine quantitatively the interference with conjugation of cell-wall fragments from both male and female strains of Escherichia coli. The fertility of the donor is reflected in the assay. Phenol extracts from the cell walls were active, and chemical analysis suggested that the activity resided in the lipopolysaccharide fraction.

Cell Extracts↗

PubMed1993

Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross-linking of long and short single-stranded DNA molecules.

Band shift and UV cross-linking assays were used to analyze the major single-stranded DNA (ssDNA) binding activity in lysates of primate and rodent cells. The ssDNA binding activity behaved chromatographically similar to that of replication protein A (RP-A), a multisubunit protein containing three polypeptides of molecular mass 70, 34, and 14 kDa. A 70-kDa protein was found to harbor the ssDNA binding activity when UV cross-linked to long ssDNA or to oligonucleotide probes. Monoclonal antibodies against the 70- and the 34-kDa subunits produced super-gel-shift patterns, demonstrating that the reactive protein is indeed RP-A and that the retarded native binding complex included both subunits. RP-A displayed oligonucleotide-specific binding dependent on oligomer length. Increasing oligonucleotide length led to the formation of slow migrating complexes harboring multiple RP-A molecules, suggesting that an interval of about 20-30 bases is required for the binding of RP-A molecules. While similar binding activity was detected in cell extracts derived from proliferating and quiescent cells, a sharp decline in ssDNA binding activity was observed in the SV40-transformed Chinese hamster cell line 631 following UV irradiation. The nature of this decrease in activity and its possible effect on DNA replication is discussed.

Animals↗

PubMed1991

Nucleotide excision repair of DNA by human cell extracts is suppressed in reconstituted nucleosomes.

Nucleotide excision repair is a complex biochemical process that corrects DNA damage caused by a broad spectrum of physical and chemical agents. We examined the effect of the assembly of ultraviolet-irradiated plasmid DNA into nucleosomes on nucleotide excision repair supported by human cell extracts. Repair synthesis in unassembled UV-irradiated plasmid DNA was readily detected in extracts from repair-proficient human cells. In contrast, repair synthesis was markedly suppressed in UV-irradiated DNA assembled into nucleosomes (minichromosomes). This suppression occurred at a step(s) which precedes repair synthesis during nucleotide excision repair. Human cell extracts were unable to effectively assemble plasmid DNA into nucleosomes under repair synthesis conditions. The addition of purified histones to the extracts restored their capacity for nucleosome assembly and simultaneously led to the suppression of repair synthesis. We propose that the preferential repair of actively transcribed genes relative to transcriptionally silent genes may reflect altered nucleosome conformation during transcription.

Animals↗

PubMed1984

Use of a 2-5A analogue probe for detecting RNA ligase and RNA ligase substrates in mammalian cell extracts.

The compound ppp(A2'p)3A3'[32P]pCp is a commercially available radioactive analogue of the 2',5' oligoadenylate series ppp(A2'p)nA, n greater than or equal to 2, commonly referred to as 2-5A. It is used as a probe for measuring concentrations in competition radiobinding and radioimmune assays. We have found that incubation of the probe with extracts from HeLa, CV1, or neuroblastoma cells results in its covalent attachment to two size classes of RNA: the first includes a major species with a molecular weight of approximately 350,000, the second is much smaller (40 +/- 5 nucleotides in length) and could represent tRNA half-molecules. Ligation is to the 3' end of the probe molecule with formation of a 3',5'-phosphodiester bond. Thus, probe ligation provides a sensitive and convenient assay for the detection not only of RNA ligase(s) but also of ligatable RNAs (such as the putative tRNA half-molecules) in mammalian cell extracts.

Animals↗

PubMed1983

Regulation of bile acid 7-dehydroxylase activity by NAD+ and NADH in cell extracts of Eubacterium species V.P.I. 12708.

The 7 alpha-dehydroxylation of primary bile acids by Eubacterium sp. V.P.I. 12708 required a cell extract prepared from a cholic acid-induced culture and NAD+. NADH (0.5 mM) inhibited bile acid 7-dehydroxylase activity more than 50% when added to reaction mixtures containing NAD+ (0.5 mM). Saturation kinetics and double reciprocal plots of NADH inhibition were consistent with negative cooperativity. 7-Dehydroxylase activity was modulated by the molar ratio of NAD+-NADH with maximal activity at a NAD+ mole fraction of 0.75 to 0.85. NADH stimulated 7-dehydroxylase activity (30% to 50%) at low concentration (less than 0.15 mM) and inhibited at higher concentrations. Reduction of the proposed delta 6-intermediate (3 alpha-hydroxy-5 beta-6-cholen-24-oic acid) to lithocholic acid required a cell extract from a cholic acid-induced culture and was stimulated by the addition of NAD+. Reduced flavin nucleotides stimulated (32% to 62%) and NADH (0.5 mM) inhibited (78%) the reduction of the delta 6-intermediate to lithocholic acid. 7-Dehydroxylase was highly specific for bile acid substrates and required a free C-24 carboxyl group and an unhindered 7 alpha- or 7 beta-hydroxy group on the B-ring of the steroid nucleus for activity. Bile acid 7 alpha- and 7 beta-dehydroxylase and delta 6-reductase activities all co-eluted from an anaerobic high performance liquid chromatography gel filtration column. However, approximately 80% to 96% of the total units of activity were lost. A substantial portion (20% to 30%) of the total activity was recovered when material from low molecular weight (8,000 to 14,000 Mr) eluting fractions was added back to fractions containing enzyme activity. These studies show that 7-dehydroxylase is highly specific for substrates and its activity may be regulated by the NAD+-NADH ratio in the bacterial cell.

Carbon Radioisotopes↗

PubMed2005

Competing roles of cytochrome P450 1A1/1B1 and aldo-keto reductase 1A1 in the metabolic activation of (+/-)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene in human bronchoalveolar cell extracts.

(+/-)-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol), a proximate carcinogen derived from benzo[a]pyrene (BP) requires further metabolic activation to exert its carcinogenic effects. Two principal pathways have been implicated, and these involve either the formation of (+/-)-trans-7,8-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) catalyzed by P450 1A1/P450 1B1 (NADPH-dependent monoxygenases) or the formation of benzo[a]pyrene-7,8-dione (BP-7,8-dione) catalyzed by human aldo-keto reductases AKR1A1 and AKR1C1-AKR1C4 [NAD(P)(H)-dependent oxidoreductases]. The relative contributions of the two pathways to PAH activation are unknown. In this study, BP-7,8-diol metabolism was studied in human bronchoalveolar H358 cell extracts. Parental H358 cells do not constitutively express P450 1A1/P450 1B1 or AKRs but were manipulated by induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to express P450 1A1/P450 1B1 or by stable transfection to express AKR1A1 (aldehyde reductase). TCDD induction of AKR1A1 transfectants provided a cell line that expressed both pathways. Extracts derived from parental H358 cells plus TCDD (P450 induction) produced electrophilic anti-BPDE, which hydrolyzed to benzo[a]pyrene tetrahydrotetrols (BP-tetrols), extracts derived from AKR1A1-transfected cells (AKR1A1 expression) produced reactive and redox-active BP-7,8-dione, which was trapped in situ as its mono(thioether) conjugate, and extracts derived from AKR1A1 transfectants plus TCDD (coexpression of P450 1A1/P450 1B1 and AKR1A1) produced both anti-BPDE and BP-7,8-dione. The competing activation of BP-7,8-diol by P450 1A1/P450 1B1 and AKR1A1 was studied with varied NADPH:NAD+ ratios. The system with a relatively higher concentration of NADPH favored formation of anti-BPDE via P450 1A1/P450 1B1, while the system with the higher concentration of NAD+ favored formation of BP-7,8-dione via AKR1A1. Under conditions that mimic the cellular redox state, 10 microM NADPH and 1 mM NAD+, equal amounts of BP-tetrols and BP-7,8-dione were formed. This suggests that P450 1A1/P450 1B1 and AKR1A1 play competing roles in the metabolic activation of BP-7,8-diol and that the dominant pathway of BP-7,8-diol activation depends on the redox state of the cells. These model systems provide a cellular context in which the dominant DNA adducts/lesions formed by either pathway may be compared.

Alcohol Oxidoreductases↗

PubMed2004

Reversion of protein aggregation mediated by Sso7d in cell extracts of Sulfolobus solfataricus.

In eukaryotic cells and in Escherichia coli, reversion of protein aggregation is mediated by the network of chaperones belonging to Hsp70 and Hsp100 families [Weibezahn, Bukau and Mogk (2004) Microb. Cell Fact. 3, 1-12]. The thermophilic prokaryotes of the archaea domain lack homologues of these chaperone families, and the mechanisms they use to rescue aggregated proteins are unknown [Macario, Malz and Conway de Macario (2004) Front. Biosci. 9, 1318-1332]. In the present study, we show that stable protein aggregates can be detected in extracts of starved cells of the thermophilic archaeon Sulfolobus solfataricus, and that the protein Sso7d interacts with the aggregates and mediates the disassembly of the aggregates and the re-activation of insolubilized beta-glycosidase in the presence of ATP hydrolysis. Furthermore, we report that heat-induced protein aggregates in extracts of exponential cells of S. solfataricus contain Sso7d that rescues insolubilized proteins in the presence of ATP hydrolysis. Results of these experiments performed in cell extracts are consistent with an in vivo role of Sso7d in reverting protein aggregation.

Adenosine Triphosphate↗

PubMed1998

Cytokine profiles of cells extracted from humans with periodontal diseases.

FACS analysis was used to determine the percent interferon (IFN)-gamma-, interleukin (IL)-4-, IL-10-, and CD30-positive T-cells extracted from adult periodontitis (AP) and healthy/gingivitis (H/G) subjects. Additionally, the percentages of IL-1 beta-, IL-10- and IL-12-producing B-cells and macrophages were ascertained. The percent IL-10+ CD8 cells extracted from AP lesions was decreased compared with H/G tissues (p = 0.033), and the percent CD30+ CD4 (p = 0.001) and CD30+ CD8 (p = 0.028) cells was higher in AP than in H/G tissues. The percentages of IL-1 beta + macrophages (p = 0.003) and IL-12+ B-cells (p = 0.034) were both higher in AP lesions than in H/G tissues. The specific effect of Porphyromonas gingivalis on the cytokine profiles of peripheral blood mononuclear cells isolated from P. gingivalis-infected AP and H/G patients was also determined. While there were no significant differences in the percent cytokine-positive T-cells after stimulation with P. gingivalis outer membrane antigens (OM) for 6 days compared with cells incubated in medium only, the percent CD30+ CD4 cells increased significantly (p = 0.047 and p = 0.063 for AP and H/G groups, respectively). There was also an increase in the percent IL-1 beta + B-cells from AP patients (p = 0.029), and the percent IL-12+ monocytes from AP and H/G subjects was higher than the percent IL-12+ B-cells, both after stimulation with P. gingivalis OM (p = 0.005 for the AP group and p = 0.058 and therefore not quite significant for the H/G group) and when incubated in medium alone (p = 0.016 and p = 0.015 for AP and H/G groups, respectively). This study has shown that IL-10+ CD8 cells may be significant in gingival lesions, and that CD30+ T-cells indicative of Th2 or Th0 cells may play a role in progressive periodontal disease. This study has also shown that B-cells produce IL-1 in the gingival lesion and that P. gingivalis may be significant in the induction of B-cell-induced IL-1.

Adult↗