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PubMed1991

Hydrolysis and reduction of factor 390 by cell extracts of Methanobacterium thermoautotrophicum (strain delta H).

Cell extracts of Methanobacterium thermoautotrophicum (strain delta H) were found to perform a hydrogen-dependent reduction of factor 390 (F390), the 8-adenylyl derivative of coenzyme F420. Upon resolution of cell extracts, F390-reducing activity copurified with the coenzyme F420-dependent hydrogenase. This indicates that F390 serves as a substrate of that enzyme. Activity towards F390 was approximately 40-fold lower than that towards coenzyme F420 (0.12 and 5.2 mumol.min-1.mg of protein-1, respectively). In addition, cell extracts catalyzed the hydrolysis of F390 to AMP and coenzyme F420. This hydrolysis required the presence of thiols (6 mM) and much ionic strength (1 M KCl) and was reversibly inhibited by oxygen. The reaction proceeded optimally at pH 8.2 and was Mn dependent. Conditions for F390 hydrolysis in cell extracts are in many respects opposite to those previously described for F390 synthesis.

Adenosine Monophosphate↗

PubMed1988

Antibody production against BLV-p24 in calves following application of cell extracts from tumorous lymph nodes of cattle with enzootic bovine leukosis.

In the cell extract from tumorous lymph nodes of bovine leukosis virus (BLV)-infected cattle (tumour cell extract) and from lymph nodes of BLV-free cattle (control cell extract) neither the gp51 nor the p24 antigens were detectable. The tumour cell extract contained receptors for the BLV antigens gp51 and p24. The immunogenicity of the cell extracts was tested in calves. All calves treated with the tumour cell extract developed antibodies against p24 but not against gp51. Administration of the control cell extract, in contrast, induced no antibodies against p24.

Animals↗

PubMed1982

7 beta-Dehydroxylation of ursodeoxycholic acid by whole cells and cell extracts of the intestinal anaerobic bacterium, Eubacterium species V.P.I. 12708.

Whole cells and cell extracts of Eubacterium species V. P. I. 12708 7-dehydroxylated [3H]ursodeoxycholic acid or [14C]chenodeoxycholic forming lithocholic acid. 7 beta-Dehydroxylation specific activity was 146 and 386 nmol hr-1 mg protein-1 for cell extracts and whole cells, respectively. 7 alpha- or 7 beta-Dehydroxylation activity was detected only in whole cells or cell extracts prepared from cultures grown in the presence of cholic acid. The addition of NAD+ (0.5 mM) to anaerobically dialyzed cell extracts stimulated 7 beta- and 7 alpha-dehydroxylation activity by 5- and 40-fold, respectively. The level of 7 beta-dehydroxylation specific activity was approximately 3- to 5-fold lower than 7 alpha-dehydroxylation in whole cells and 3-fold lower in cell extracts. Substrate saturation kinetics for ursodeoxycholic acid and chenodeoxycholic acid were hyperbolic and showed substrate inhibition at concentrations above 200 microM. The apparent Km values for ursodeoxycholic and chenodeoxycholic acid were 14.5 microM and 49 microM, respectively. Both 7 alpha- and 7 beta-dehydroxylase activities were inactivated (60% to 70%) by heating for 6 min at 45 degrees C. Moreover, both activities co-eluted from a anaerobic Bio-Gel A 1.5-M column as a single peak at approximately 114,000 (Mr). These data show that this intestinal anaerobic bacterium has both 7 alpha- and 7 beta-dehydroxylase activities which may be catalyzed by the same enzyme.U

Chenodeoxycholic Acid↗

PubMed1993

O6-methylguanine residues elicit DNA repair synthesis by human cell extracts.

We have investigated the processing of O6-methylguanine (O6-MeGua) in plasmid DNA by extracts of human cells defective in O6-MeGua-DNA methyltransferase. Cell extracts of HeLaMR cells performed viral T antigen-independent DNA synthesis on plasmids that had been treated with low concentrations of methylating agents. The in vitro DNA synthesis was non-semiconservative and depended on the presence of O6-MeGua in the substrate. The involvement of DNA polymerase delta or epsilon and proliferating cell nuclear antigen but not single-strand binding protein was indicated by partial fractionation, inhibitor, and antibody studies. Processing of O6-MeGua is not via the UV nucleotide excision repair pathway since additional component(s) are apparently required to perform repair synthesis on the methylated substrate. This is the first direct demonstration of DNA repair synthesis provoked by O6-MeGua in DNA. Since O6-MeGua is not excised from DNA by Mex- cells, it represents a novel type of processing of the methylated base that may be involved in its cytotoxicity.

Antibodies↗

PubMed1990

Pyrogallol-to-phloroglucinol conversion and other hydroxyl-transfer reactions catalyzed by cell extracts of Pelobacter acidigallici.

Permeabilized cells and cell extracts of Pelobacter acidigallici catalyzed the conversion of pyrogallol (1,2,3-trihydroxybenzene) to phloroglucinol (1,3,5-trihydroxybenzene) in the presence of 1,2,3,5-tetrahydroxybenzene. Pyrogallol consumption by resting cells stopped after lysis by French press or mild detergent (cetyltrimethylammonium bromide [CTAB]) treatment. Addition of 1,2,3,5-tetrahydroxybenzene to the assay mixture restored pyrogallol consumption and led to stoichiometric phloroglucinol accumulation. The stoichiometry of pyrogallol conversion to phloroglucinol was independent of the amount of tetrahydroxybenzene added. The tetrahydroxybenzene concentration limited the velocity of the transhydroxylation reaction, which reached a maximum at 1.5 mM tetrahydroxybenzene (1 U/mg of protein). Transhydroxylation was shown to be reversible. The equilibrium constant of the reaction was determined, and the free-energy change (delta G degree') of phloroglucinol formation from pyrogallol was calculated to be -15.5 kJ/mol. Permeabilized cells and cell extracts also catalyzed the transfer of hydroxyl moieties between other hydroxylated benzenes. Tetrahydroxybenzene and hydroxyhydroquinone participated as hydroxyl donors and as hydroxyl acceptors in the reaction, whereas pyrogallol, resorcinol, and phloroglucinol were hydroxylated by both donors. A novel mechanism deduced from these data involves intermolecular transfer of the hydroxyl moiety from the cosubstrate (1,2,3,5-tetrahydroxybenzene) to the substrate (pyrogallol), thus forming the product (phloroglucinol) and regenerating the cosubstrate.

Bacteria, Anaerobic↗

PubMed1991

In vitro assembly of poliovirus 14 S subunits: identification of the assembly promoting activity of infected cell extracts.

Purified poliovirus 14 S subunits are assembled into empty capsids in vitro only if their concentration exceeds a 1.6 nM threshold. This also holds true for the 14 S subunits in unpurified extracts of infected cells. Such an extract may promote the assembly of extraneous 14 S subunits, but only if it contributes enough 14 S subunits to raise their total concentration over the threshold. As a result of assembly, the concentration of 14 S subunits in an infected cell extract decreases exponentially, with a half life of 15 min at 37 degrees. When purified 14 S subunits of serotype 1 are mixed with extracts of cells infected with type 2 or 3, chimeric empty capsids are formed, thus showing the pooling of endogenous and extraneous 14 S subunits. In conclusion, the assembly promoting activity of infected cell extracts amounts to nothing more than the supply of endogenous 14 S subunits.

Antibodies, Monoclonal↗

PubMed1994

DNA loop repair by human cell extracts.

An activity in human cell extracts is described that repairs DNA with loops of five or more unpaired bases. Repair is strand-specific and is directed by a nick located 5' or 3' to the loop. This repair is observed in a colorectal cancer cell line that is devoid of a wild-type hMLH1 gene and is deficient in repair of mismatches. However, a cell line with deletions in both hMSH2 alleles is deficient in repair of both loops and mismatches. Defects in loop repair may be relevant to the repetitive-sequence instability observed in cancers and other hereditary diseases.

Adaptor Proteins, Signal Transducing↗

PubMed1989

Activation of formylmethanofuran synthesis in cell extracts of Methanobacterium thermoautotrophicum.

In cell extracts of Methanobacterium thermoautotrophicum, formylmethanofuran (formyl-MFR) synthesis (an essential CO2 fixation reaction that is an early step in CO2 reduction to methane) is subject to a complex activation that involves a heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine O3-phosphate (CoM-S-S-HTP). In this paper we report that titanium(III) citrate, a low-potential reducing agent, stimulated CO2 reduction to methane and activated formyl-MFR synthesis in cell extracts. Titanium(III) citrate functioned as the sole source of electrons for formyl-MFR synthesis and enabled this reaction to occur independently of CoM-S-S-HTP. In addition, CoM-S-S-HTP was found to activate an unknown electron carrier that reduced metronidazole. The activation of formyl-MFR synthesis by CoM-S-S-HTP may involve the activation of a low-potential electron carrier.

Cell-Free System↗

PubMed1990

Restoration of the responsiveness to growth factors in senescent cells by an embryonic cell extract.

In senescent fibroblast cell cultures which have approached a postmitotic stage in vitro, responsiveness to growth factors is restored upon exposure to an embryonic sheep cell extract. The extract contains molecules below a molecular weight of 1 x 10(5) Da in aqueous solution. Following a transient exposure to the extract, mitotic activity is resumed, and the cells keep dividing over several passages. The target cells which respond to the treatment were identified in a single-cell assay as those that still had the capacity to undergo at least several mitotic divisions before entering the final stage of senescence.

Animals↗

PubMed1991

Effect of leukaemic sera & cell-extracts on splenic colony counts (CFU-S).

Sera and leukaemic cell extracts from patients of acute leukaemia were evaluated for their effect on the repopulating ability of the pluripotent stem cells and erythroid differentiation by an in vivo splenic colony count (CFU-S) technique. Normal donor marrow cells of mice were treated with sera and cell extracts from patients of acute leukaemic and healthy controls and injected in the recipient mice. The CFU-S performed on the seventh day to assess repopulating ability of the stem cell showed consistently lower CFU-S counts in the test groups, with leukaemic sera (P less than 0.01) as well as leukaemic cell-extracts (P less than 0.001). The erythroid differentiation assessed by 59Fe uptake by the spleens also showed significantly reduced counts in the two test groups (P less than 0.01 and less than 0.001 respectively). The results indicate that both leukaemic sera and cell-extracts exert a significant suppressive effect on the repopulating ability of the stem cells and on their erythroid differentiation.

Acute Disease↗

PubMed1985

Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. wt. heat-shock protein in animal cell extracts.

Double-stranded DNA (dsDNA) induces the transfer of phosphate from ATP to several proteins in extracts of widely divergent eukaryotic cells. Extracts of HeLa cells, rabbit reticulocytes, Xenopus eggs and Arbacia eggs all show dsDNA-dependent protein phosphorylation. The mechanism is specific for dsDNA and will not respond to either RNA or single-stranded DNA. One of the proteins which is phosphorylated in response to dsDNA has a subunit mol. wt. of 90 000 and has been identified as a heat-shock protein (hsp90). Although mouse cell extracts were shown to contain hsp90, they failed to show a dsDNA-dependent protein phosphorylation. The observation that dsDNA can modulate the phosphorylation of a set of proteins raises the possibility that dsDNA may play a role as a cellular regulatory signal.

Animals↗

PubMed1990

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS).

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol min-1 mg-1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridium pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 M. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.

Acetates↗

PubMed1982

In vitro effect of endotoxin from Shigella sonnei. phase I on human blood platelets and mononuclear leucocytes: comparison of "free endotoxin" with cell-extracted preparations.

The in vitro effect of a purified endotoxin preparation from culture fluids of Shigella sonnei, phase I (purified free endotoxin, PFE) and of three endotoxin preparations chemically extracted from the intact parent cells on human blood mononuclear leucocytes and platelets was investigated. PFE, like cell-extracted preparations, caused generation of strong procoagulant activity (tissue factor) by human mononuclear cells. PFE-stimulated cells, however, developed significantly greater activity than cells stimulated by the other endotoxins. they had about 4-fold more activity. Neither free nor cell-extracted preparations induced aggregation in human citrated or heparinized platelet-rich plasma (PRP) or unmasking of platelet factor 3 (PF3). These findings suggest that free endotoxin from Shigella sonnei, phase I resembles endotoxin extracted from cells by conventional procedures in their interaction with human platelets and mononuclear leucocytes. In view of the possible contribution of free endotoxin to endotoxemia in human and experimental gram-negative sepsis, our data that free endotoxin stimulates human mononuclear leucocytes to produce a potent trigger of blood coagulation (tissue factor) may be relevant to the understanding of the mechanism(s) responsible for the initiation of intravascular coagulation in severe human infections.

Blood Platelets↗

PubMedDate not supplied

Activation of autologous or HLA-identical sibling cytotoxic T lymphocytes by blood derived dendritic cells pulsed with tumor cell extracts.

This study describes the effect of dendritic cells (DC) pulsed with tumor cell extracts on the induction and activation of autologous and HLA-identical sibling tumor cell specific cytotoxic T lymphocytes (CTL). The subjects of the study were patients with hematological malignancies (acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma), and stem cell transplant donors. Although lymphocytes primed with non-pulsed DC did not show definite cytotoxic activity against autologous or HLA-identical sibling patient tumor cells, both autologous and HLA-identical sibling CTL activated by DC pulsed with tumor cell extracts demonstrated markedly significant killing activity against tumor cells in the fashion of higher cytotoxicity with the increase of effector:target ratio (p<0.05). These findings reveal that DC pulsed with tumor cell extracts could be efficiently applicable in anti-tumor immunotherapy for patients with hematological malignancies.

Adult↗

PubMed1990

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of "Desulfomonile tiedjei".

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, "Desulfomonile tiedjei." We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c(3), or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H(2), but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35 degrees C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in "D. tiedjei" cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in "D. tiedjei" extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.

Journal Article↗

PubMed1993

Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. I. Development of a cell-free system detecting excision repair of UV-irradiated SV40 chromosomes.

Simian virus 40 minichromosomes were irradiated with ultraviolet light and used to study cell-free DNA repair reactions by soluble extracts of repair-proficient human cells. Damage-specific incorporation of labeled nucleotides was measured after linearization and electrophoresis of the viral DNA. Repair synthesis with irradiated chromosomes was dependent on the dose of ultraviolet light, but naked viral DNA irradiated at the same doses showed significantly higher template activity. The reactions absolutely required the presence of cell extract, but an extract from Chinese hamster cells was as active as those from human cells. The repair activity was apparently stimulated by the addition of unirradiated plasmid DNA, which did not affect the nucleosome density of the viral chromosomes. ATP and its regenerating system were required for the reaction, but total repair synthesis was little affected by exogenous addition of substrates for RNA synthesis and poly(ADP-ribosyl)ation. Most of the repaired viral chromosomal DNA was in the supercoiled, closed circular form, suggesting that excision repair reactions proceeded to the stage of ligation in the present cell-free system.

Animals↗

PubMed1986

Presence of an insulin-stimulated serine kinase in cell extracts from IM-9 cells.

Insulin responsive protein kinase activities of wheat germ purified glycoproteins were examined. Glycoproteins were first incubated without or with insulin, and then exposed to a serum containing antibodies to insulin receptor. Thereafter, both immunoprecipitates and supernatants were studied for their kinase activity toward histone. Incubation with anti receptor antibodies promoted insulin receptor beta subunit and histone phosphorylation. More important insulin receptor depleted extract contained a kinase activity toward histone, that was increased by preincubation with insulin. This stimulation was observed only when insulin was added before the immunoprecipitation of insulin receptors. Alkali treatment and phosphoamino acids analysis revealed that the kinase activity remaining in the supernatant is serine specific. These findings suggest, that a serine kinase activity is associated with the insulin receptor, that it can be separated from the insulin receptor with anti receptor antibodies, that the serine kinase is activated by the hormone-receptor complex.

Cell Line↗

PubMed1991

Formation of factor 390 by cell extracts of Methanosarcina barkeri.

Cell extracts of Methanosarcina barkeri converted coenzyme F420 in an ATP-dependent reaction to the adenylylated derivative factor 390. Although it was reported previously (L. M. Gloss and R. P. Hausinger, BioFactors 1:237-240, 1988) that whole cells were unable to perform this conversion, we observed the conversion in 7 of 11 extracts, all of which were prepared from different batches of cells.

Adenosine Monophosphate↗