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At least 55 records · Page 3Linked to original sources
PubMed1974

Reciprocal inhibition of mouse leukemia virus infection by Fv-1 allele cell extracts.

Soluble extracts of mouse cells with Fv-1(n) or Fv-1(b) gene alleles specifically and reciprocally inhibit infection of B- or N-tropic mouse leukemia viruses in permissive cell cultures. NB-tropic virus infection was not inhibited by either cell extract. Extracts from Fv-1(-) cells did not inhibit infection by the three virus host-range types, but N- or B-tropic virus infection of Fv-1(-) cells was inhibited by extracts of the nonpermissive cells, and Fv-1(nb) cell extracts inhibited both viruses. The maximum degree of inhibition was 50-80% as determined by immunofluorescent or plaque assays, with extracts containing up to 500 mug/ml of nonpermissive cell protein. The inhibitor(s) is relatively unstable since activity is lost after 2 hr at 37 degrees or 30 min at 56 degrees . The inhibitor(s) was most effective if added 2 hr before or within 2 hr after infection, did not react with the virus directly, inhibit virus attachment, or inhibit the normal cell functions tested. These results indicate that nonpermissive mouse cells contain a product, possibly determined by the Fv-1 gene, which inhibits some early postpenetration event(s) in leukemia virus infection.

Alleles↗
PubMed2001

DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts.

In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.

Amino Acid Substitution↗
PubMed2003

Monitoring liposome-mediated delivery and fate of an antisense drug in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used for evaluation of the effectiveness of delivery and fate of a model 25-mer DNA-based phosphorothiate antisense drug in cells. The antisense molecule was delivered to the cells through a simple incubation and by using a cationic liposome (Cytofectin GS 3815). The cationic lipid interacted with the negatively charged antisense to form a more positively charged complex. It was observed that uptake of the liposome-antisense complex by the cell was dependent on concentration of lipid, duration of transfection, and the cell type. The antisense drug interacted with intracellular components such as proteins and additional steps were needed to quantify the free antisense. Proteinase-K was able to release antisense from proteins. However, the addition of sodium dodecyl sulfate (SDS) to the sample or running buffer was more effective than Proteinase-K to release both naked and liposome-bound antisense from the cellular materials. Analysis of single HeLa cells for uptake of the unbound and liposome-complexed antisense revealed averages of 8.9x10(-19) moles and 4.9x10(-18) moles, respectively. The amount of uptake, however, varied greatly among individual cells and depended on the delivery method. With liposome-mediated delivery, the relative standard deviation (RSD) for the amount of antisense in individual cells was 130%, while the variation was much smaller (RSD = 45%) when the cells were incubated with the unbound antisense. These uptake variations agreed with those obtained from flow cytometry analysis.

Cell Extracts↗
PubMed1983

Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts.

Using a whole cell extract of HeLa cells, we synthesized unspliced RNAs containing the first two leaders and the first intervening sequence of the adenovirus 2 major late transcription unit. Upon incubation of these pre-mRNAs in reaction mixtures containing a nuclear extract and a postnuclear fraction (S100), removal of the first intervening sequence and concomitant joining of the first leader to the second leader was observed. This splicing reaction requires proteins, Mg2+ ions, and ATP. The S100 fraction alone has no splicing activity but stimulates splicing when added to the nuclear extract. Upon fractionation of the postnuclear S100 by chromatography on ion exchange and gel filtration columns, the stimulatory activity copurifies with small ribonucleoprotein particles.

Adenosine Triphosphate↗
PubMed1985

Methenyl-tetrahydromethanopterin cyclohydrolase in cell extracts of Methanobacterium.

Cell extracts of Methanobacterium thermoautotrophicum possess a methenyl-tetrahydromethanopterin (methenyl-H4MPT) cyclohydrolase. The enzyme catalyzes the hydrolysis of methenyl-H4MPT to formyltetrahydromethanopterin (formyl-H4MPT). The reaction is reversible and both the rate and extent of the reaction depend on the pH and the buffer used. Similarly, the nonenzymatic hydrolysis of methenyl-H4MPT is highly dependent on pH and buffer. An active derivative of methenyl-H4MPT was obtained in 94% yield by reacting H4MPT with formic acid in the presence of excess acetic acid under anoxic conditions at 80 degrees C for 3 h. H NMR spectroscopy and fast atom bombardment mass spectrometry revealed the product to be a derivative of methenyl-H4MPT which had lost the alpha-hydroxyglutarylphosphate unit. In spite of this loss, this derivative served both as a substrate for methanogenesis and for the cyclohydrolase. Comparison of the properties of the products of the enzymatic and nonenzymatic hydrolyses indicates that the enzymatic reaction yields N5-formyl-H4MPT whereas the nonenzymatic reaction yields N10-formyl-H4MPT.

Aminohydrolases↗
PubMed2002

Repair of dihydrouracil supported by base excision repair in mNTH1 knock-out cell extracts.

In mammalian cells, thymine glycols and other oxidized pyrimidines such as 5,6-dihydrouracil are removed from DNA by the NTH1 protein, a bifunctional DNA-N-glycosylase. However, mNTH1 knock-out mice in common with other DNA glycosylase-deficient mice do not show any severe abnormalities associated with accumulation of DNA damage and mutations. In the present study we used an in vitro repair system to investigate the mechanism for the removal of 5,6-dihydrouracil from DNA by mNTH1-deficient cell-free extracts derived from testes of mNTH1 knock-out mice. We found that these extracts are able to support the removal of 5,6-dihydrouracil from DNA at about 20% of the efficiency of normal extracts. Furthermore, we also found that single-nucleotide patch base excision repair is the major pathway for removal of 5,6-dihydrouracil in mNTH1-deficient cell extracts, suggesting the involvement of other DNA glycosylase(s) in the removal of oxidized pyrimidines.

Animals↗
PubMed1985

Characterization of delta 4-3-ketosteroid-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase in cell extracts of Clostridium innocuum.

Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-ketosteroids to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively. delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity. 5 beta-Reductase from C. innocuum had a pH optimum of 5.0. The substrate concentration at half-maximal reaction velocity was 4.2 microM, and a specific activity of 17 nmol product formed/h per mg protein was determined using 4-pregnen-3,20-dione (progesterone) as a substrate. delta 4-3-Ketosteroid-5 beta-reductase from C. innocuum reduced progesterone and testosterone, but not 4-cholesten-3-one, to corresponding 3-keto-5 beta derivatives. A relative molecular (Mr) weight of 80 000 was estimated for 5 beta-reductase using HPLC-gel filtration chromatography. 3 beta-Hydroxysteroid dehydrogenase in cell extracts of C. innocuum was oxygen sensitive and required NADH for activity. An Mr of 80 000 was estimated for 3 beta-hydroxysteroid dehydrogenase. However, 5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase activities were separated using an HPLC-DEAE chromatography technique.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

Effects of growth rate on cell extract performance in cell-free protein synthesis.

Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity.

Cell-Free System↗
PubMed1979

Grouping of lactic streptococci by gel electrophoresis of soluble cell extracts.

Soluble cell proteins obtained from 35 strains of lactic streptococci were examined by gel electrophoresis. A mathematical analysis of the densitometer scans of the gels enabled strains to be grouped according to their overall similarity. Strains which were known to be variants of the same parent strain fell into the same group, supporting the validity of the method. It is suggested that strains which are alike according to their gel electrophoretic patterns when grown under standard conditions have an overall phenotypic similarity and that this indicates a similarity in genotype. The relevance of this to selection of strains of lactic streptococci for cheesemaking is discussed.

Journal Article↗
PubMed2002

Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 microM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635 +/- 27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61 +/- 13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid-liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid-liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.

Antibiotics, Antineoplastic↗
PubMed1993

Repair and misrepair of site-specific DNA double-strand breaks by human cell extracts.

The rejoining by human cell extracts of a double-strand break induced by endonuclease treatment at one of several sites within a small DNA molecule was studied. Rejoining was found at each of 8 sites tested, but the rejoin efficiency varied with the nature of the break (e.g., breaks with cohesive ends were rejoined more efficiently than blunt-ended breaks). Extracts from primary and immortalized cell lines, as well as those from individuals with ataxia telangiectasia (A-T), showed the same pattern of relative rejoin efficiencies. However, mis-rejoining varied with the cell extract used, and was particularly elevated with two immortalized A-T cell lines. Mixing experiments showed that the mis-rejoining property of extracts could act in a semi-dominant fashion, depending on the individual efficiencies of the component extracts. The mis-rejoin mechanism involved deletion at sites of short direct repeats at various distances from the initial break site. A model of deletion formation (the strand-exposure and repair model) is restated to explain the sequence repeat dependence found, and is compared to models of homologous DNA recombination.

Ataxia Telangiectasia↗
PubMed1991

Properties of a 4-ene-3-ketosteroid-5 alpha-reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144.

When Eubacterium sp. 144 was grown in the presence of progesterone, extracts of these cells contained a 4-ene-3-ketosteroid-5 alpha-reductase (5 alpha-reductase). No evidence for the presence of a 5 beta-steroid-reductase or a 5 alpha to 5 beta-steroid-isomerase was found. 5 alpha-Reductase activity was dependent on reduced methyl viologen as the electron donor and this could be generated biologically by adding pyruvate or H2 to cell extracts or chemically by adding sodium dithionite. NADH or NADPH with or without flavin nucleotides were not electron donors for 5 alpha-reductase. Most of the 5 alpha-reductase activity (60-65%) of crude extracts was located in the membrane fraction and the enzyme was solubilized by treatment with 1% Triton X-100. Optimum 5 alpha-reductase activity occurred at pH 7.0-7.5 in potassium phosphate buffer but was stimulated by Tris-HCl buffer (pH 8.0-9.0). 5 alpha-Reductase activity was highest at 10% (v/v) methanol and was progressively inhibited by higher methanol concentrations. Sulfhydryl reagents strongly inhibited 5 alpha-reductase but the enzyme was not affected by other metabolic inhibitors. Extracts prepared from cells induced with 16-dehydroprogesterone and grown without hemin contained 5 alpha-reductase and 16-dehydroprogesterone-reductase activities equivalent to those found in extracts of induced cells grown with hemin. This indicates that hemin is not required for the synthesis of active steroid double bond-reductases in strain 144.

Cholestenone 5 alpha-Reductase↗
PubMed1990

Sexual response of Saccharomyces cerevisiae: phosphorylation of yeast glyoxalase I by a cell extract of mating factor-treated cells.

The phosphorylation of glyoxalase I was observed when the phosphatase-treated enzyme was incubated in the presence of [gamma-32P]ATP and a cell extract prepared from alpha-type yeast cells which had been treated with the culture supernatant of a-type yeast cells. The phosphorylated protein was identified as glyoxalase I by using anti-glyoxalase I rabbit immunoglobulin G.

Basidiomycota↗
PubMed1993

Replication of UV-irradiated DNA in human cell extracts: evidence for mutagenic bypass of pyrimidine dimers.

We have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the alpha-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2 DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli alpha-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C-->T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC-->TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer.

DNA↗
PubMed2002

Nonradioactive technique to measure protein phosphatase 2A-like activity and its inhibition by drugs in cell extracts.

A nonradioactive assay has been developed that can be used to measure serine/threonine protein phosphatase (PP) activity, especially PP2A, in crude extracts from different cell lines. For this technique commercially available casein is used as an already phosphorylated substrate. The prerequisite for reliable measurements is the removal of free phosphate from cell extracts and substrate preparations with desalting columns. The use of different nonspecific or specific inhibitors as well as inhibition characteristics observed after extract dilution suggests that in the absence of magnesium, PP2A-like activity in the extracts is measured by this technique. Inclusion of magnesium allowed the detection of a protein phosphatase activity that is activated by magnesium, which is presumably PP2C. The use of structurally different as well as structurally related inhibitors of PP2A gave results comparable to those of reports from the literature that were obtained with radioactive assays. Thus, our data supported our hypothesis that this nonradioactive assay can be used for the identification of newly synthesized PP inhibitors as well as for performing structure-activity analysis within groups of such new agents.

Animals↗
PubMed1987

Structure of a methanofuran derivative found in cell extracts of Methanosarcina barkeri.

Cell extracts prepared from cells of Methanosarcina barkeri grown on hydrogen and carbon dioxide, acetate, or methanol contain a coenzyme structurally related to methanofuran. This modified coenzyme was highly purified and its structure assigned as 4-[N-(gamma-L-glutamyl-gamma-L-glutamyl-gamma-L-glutamyl-gamma-L-glutamy l)-p- (beta-amino-ethyl)phenoxymethyl]-2-(aminomethyl)furan. The key structural evidence was obtained by high-resolution fast atom bombardment-mass spectrometry and 1H NMR spectroscopy. Quantitative analysis of the hydrolytic fragments of the coenzyme supported the assigned structure. We propose that this coenzyme be called methanofuran-b.

Chemical Phenomena↗
PubMed1999

13C-Isotopic enrichment of glutathione in cell extracts determined by nuclear magnetic resonance spectroscopy.

An NMR method was developed for measuring the isotopic enrichment of glutathione in extracts of cells fed a medium containing [3, 3'-13C2]cystine. Two sublines of human mammary adenocarcinoma MCF-7 cells were exposed to growth medium containing the labeled cystine for varying periods, treated with monobromobimane, harvested, and extracted with perchloric acid. The glutathione-bimane adduct was partially purified by solid-phase extraction before analysis by 1H NMR spectroscopy. The isotopic enrichment of the beta-carbon of the cysteinyl residue of glutathione was determined directly in the cell extracts without further purification. These isotopic enrichment data can be used to determine the rate of synthesis of glutathione in cell and tissue extracts.

Adenocarcinoma↗