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PubMed1975

Immunoelectrophoresis of Mycobacterium tuberculosis antigens. Comparative analysis of cell extract and culture filtrate antigens.

Immunoelectrophoretic analyses were performed on a cell extract and culture filtrate of Mycobacterium tuberculosis, H37Rv strain using homologous polyvalent goat antisera. General similarity between the cell extract and culture filtrate antigens was found. Significant variation between two anti-culture filtrate antisera was observed. These antigens and antisera also differed significantly from previously described reference reagents. These findings demonstrate the necessity of using reference materials for precise identification of mycobacterial antigens in comparative studies.

Animals↗

PubMed1998

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1985

Phosphorylation of 2-5A core 5'-diphosphate to 2-5A in mouse L cell extracts.

When added to extracts of mouse L cells containing ATP and an energy regenerating system, the 5'-diphosphate of 2-5A core, pp5'A2'p5'A2'p5'A, as well as a bromoadenylate analog, pp5' (br8A)2'p5'(br8A)2'p5'(br8A), can be phosphorylated to the corresponding 5'-triphosphate, ppp5'A2'p5'A2'p5'A and ppp5'(br8A)2'p5'(br8A)2'p5(br8A), respectively. The extent of this conversion was about 0.5% when the concentration of 5'-diphosphate was about 10(-4) M. Thus, although previous studies have shown that the 5'diphosphate, pp5'A2'p5'A2'p5'A, can activate the 2-5A-dependent endonuclease, this may be related to a phosphorylation reaction in the crude cell extracts employed in these studies and may not represent a true ability of such a 5'-diphosphate to activate directly the endonuclease.

Adenine Nucleotides↗

PubMed1988

Promoter activity of the two chicken delta-crystallin genes in a Hela cell extract.

The in vitro transcriptional activity of the two delta-crystallin genes (5'-delta 1-delta 2-3') of the chicken was studied in a whole Hela cell extract. Both the delta 1 and delta 2 promoters were recognized by RNA polymerase II in this heterologous system. The major RNA initiation site from the delta 1 promoter was the same in vitro as that which occurs in vivo, as judged by mapping with S1-nuclease, although other minor initiation sites upstream and downstream of the major initiation site were noted. A primer extension experiment showed that the longest RNA synthesized in vitro from a delta 2 template initiated near the beginning of the first exon. The delta 1 promoter was several-fold stronger than that of delta 2 under the present in vitro conditions. Transcription from the delta 1 promoter was abolished by a competitor fragment (c'-II; includes -328 to -63) purified from the delta 2 promoter, indicating that one or more common transcription factors binding upstream from the TATA box are required for in vitro function of the two delta-crystallin promoters. Thus, in the Hela cell extract both delta-crystallin genes contain a functional promoter. We consider the possibility that the single 5'CCAAT3' sequence present in the delta 1 promoter (but lacking in the delta 2 promoter) may contribute to its greater core activity under our conditions. The greater promoter activity of the delta 1-crystallin gene in the Hela cell extract was not sufficient to account for the large ratio of delta 1 to delta 2 mRNA (approximately 50 to 100) in the embryonic chicken lens.

Animals↗

PubMed2006

Mortality and toxin bioaccumulation in Bufo marinus following exposure to Cylindrospermopsis raciborskii cell extracts and live cultures.

Cylindrospermopsis raciborskii is a cyanobacterium responsible for the production of the toxin, cylindrospermopsin (CYN). Tadpoles of the cane toad Bufo marinus were exposed to freeze-thawed whole cell extracts or live cultures of C. raciborskii containing maximum CYN concentrations of 400 microg L-1 or 232 microg L-1, respectively. Exposure to live culture treatment solutions resulted in up to 66% mortality of B. marinus, whereas tadpoles exposed to whole cell extracts containing similar toxin concentrations survived. Decreases in relative growth rates and time spent for swimming were recorded from tadpoles during both types of exposure regimes. Bioconcentration of CYN was not evident following exposure to whole cell extracts containing extracellular toxin. In contrast exposure to live cultures, which contained cell-bound toxin, resulted in maximum average tissue concentrations of 895 microg free-CYN kg-1 fresh weight. This is the first investigation of C. raciborskii exposure effects and toxin bioaccumulation in the developmental stages of an amphibian.

Alkaloids↗

PubMed1975

Replication of colicin E1 plasmid DNA added to cell extracts.

Closed-circular DNA of colicin E1 plasmid can undergo a round of semiconservative replication when added to an extract of Escherichia coli. Extracts of cells that do not carry the plasmid are able to perform complete replication of the plasmid. Replication requires de novo RNA synthesis but not protein synthesis.

Bacterial Proteins↗

PubMed1990

Carbon monoxide dehydrogenase inhibitor in cell extracts of Pseudomonas carboxydovorans.

Extracts of heterotrophically grown cells of Pseudomonas carboxydovorans were found to contain an inhibitor of carbon monoxide dehydrogenase (CO-DH). The inhibitor activity was not detected in CO-autotrophically grown cells. The inhibitor was extremely stable to heat treatment based on the extent of inhibition of CO-DH activity. The extent of inhibition was proportional to the amount of cell extract added to the reaction mixture. The inhibition was independent of a prior incubation period of the extracts with CO-DH. The inhibitor was precipitable with ammonium sulfate, phenol, and trichloroacetic acid. It was passed through benzoylated dialysis tubing and Amicon ultrafiltration membrane YM2. Denaturing and nondenturing polyacrylamide gel electrophoresis of CO-DH inactivated by inhibitor revealed that the mobilities of native enzyme and subunits were identical to those of active CO-DH. The inhibitor-treated CO-DH retained its original antigenic sites and exhibited enzyme activity upon activity staining. The CO-DH inhibitor of P. carboxydovorans was also active on CO-DHs from Pseudomonas carboxydohydrogena, Acinetobacter sp. strain JC1, and Pseudomonas carboxydoflava.

Aldehyde Oxidoreductases↗

PubMed1975

Effects of red cell extract on in vitro growth and multiplication of malarial parasites.

An extract of red blood cells was prepared from monkey blood. Red cell extract (RCE) of Aotus monkeys was beneficial for the in-vitro cultivation of 2 strains of P. falciparum. The active material(s) in Aotus RCE was not sedimented by centrifugation at 34,800 g for 1 hr. Rhesus monkey RCE improved the growth of P. knowlesi for in-vitro cultivation through 4 schizogonous cycles (88 hr).

Animals↗

PubMed1993

Analysis of 1alpha,25-dihydroxyvitamin D3 receptor in cell extracts with low protein concentration.

An unacceptable loss of tritiated 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] to the wall of the reaction tubes constituted an obstacle when examining C3H/10T1/2 Cl 8 cells for 1,25-(OH)2D3 receptor. The loss of tracer in low protein cell extracts could be strongly reduced by incubating the cell extracts in polyethylene tubes and in the presence of inert peptides prepared by digestion of gluten proteins. When incubated in buffer the recovery of tracer increased from 3 to 45% by using polyethylene tubes instead of sodium-glass tubes. However, the presence of a sufficient amount of inert peptides in the buffer significantly increased the recovery of tracer to 89%. This procedure improved the saturation binding analysis of the 1,25-(OH)2D3 receptor in the C3H/10T1/2 Cl 8 cells using the hydroxylapatite assay.

Adsorption↗

PubMed1986

The mouse immunoglobulin heavy-chain enhancer: effect on transcription in vitro and binding of proteins present in HeLa and lymphoid B cell extracts.

The ability of the mouse immunoglobulin heavy chain gene (IgH) enhancer to stimulate in vitro transcription from the adenovirus-2 major late promoter (Ad2MLP) has been investigated. In agreement with the in vivo cell-type specificity of this enhancer, a stimulation can be observed in BJA-B lymphoid B cell, but not in HeLa cell, extracts. Under identical conditions, the Simian virus 40 (SV40) enhancer stimulates transcription in both extracts to approximately the same extent. In addition we have found that a sequence, previously shown to inhibit transcription in vitro in HeLa cell extracts, is also inhibitory in B cell extracts. DNase I footprint and DMS-methylation protection experiments indicate that each cell type contains proteins which bind to specific sequences of the IgH enhancer. The relationship between the binding of these proteins and the preferential activity of the IgH enhancer in B cells is discussed.

B-Lymphocytes↗

PubMed1993

An improved technique for observing both membranous organelles and cytoskeleton in saponin-extracted cells.

Detergent-extraction of soluble cytoplasmic proteins under low osmotic pressure induces severe morphological damage, particularly on membranous organelles, although this processing is often required to observe cytoskeletal elements in the cells. In the present study, the osmotic adjustment of the rinsing solution and fixative, which were used after the detergent-extraction using saponin, has greatly improved the ultrastructure of membranous organelles. This improved method allowed us to observe both cytoskeletal elements and the fine structure of membranous organelles by electron microscopy.

Animals↗

PubMed1995

Frequency and fidelity of translesion synthesis of site-specific N-2-acetylaminofluorene adducts during DNA replication in a human cell extract.

We have previously analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of SV40-based DNA replication in a human cell extract (Thomas, D. C., Veaute, X., Kunkel, T. A., and Fuchs, R. P. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7752-7756). Here we use two sets of substrates to examine the probability of replication termination and error-free and error-prone bypass of AAF adducts. The substrates contained site-specific adducts at one of three guanines in a NarI sequence (5'-GGCGCC-3') placed within the lacZ alpha reporter gene and located on the template for either leading or lagging strand replication. The presence of the adduct at any position strongly reduces the efficiency of a single round of replication in a HeLa cell extract. Product analysis reveals preferential replication of the undamaged strand and termination of replication of the damaged strand occurring one nucleotide before incorporation opposite either a leading or lagging strand adduct. Products resistant to restriction endonuclease cleavage at the adducted site were generated in amounts consistent with 16-48% lesion bypass during replication. Most of this bypass was error-free. However, two-nucleotide deletion errors were detected in the replication products of DNA containing an AAF adduct in either the leading or lagging strand, but only when present at the third guanine position. Collectively, the data suggest that the replication apparatus in a HeLa cell extract generates a template-primer slippage error at an AAF adduct once for every 30-100 bypass events.

2-Acetylaminofluorene↗

PubMed2005

Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards.

A novel method was developed for the quantitative analysis of the microbial metabolome using a mixture of fully uniformly (U) (13)C-labeled metabolites as internal standard (IS) in the metabolite extraction procedure the subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. This mixture of fully U (13)C-labeled metabolites was extracted from biomass of Saccharomyces cerevisiae cultivated in a fed-batch fermentation on fully U (13)C-labeled substrates. The obtained labeled cell extract contained, in principle, the whole yeast metabolome, allowing the quantification of any intracellular metabolite of interest in S. cerevisiae. We have applied the labeled cell extract as IS in the analysis of glycolytic and tricarboxylic acid (TCA) cycle intermediates in S. cerevisiae sampled in both steady-state and transient conditions following a glucose pulse. The use of labeled IS effectively reduced errors due to variations occurring in the analysis and sample processing. As a result, the linearity of calibration lines and the precision of measurements were significantly improved. Coextraction of the labeled cell extract with the samples also eliminates the need to perform elaborate recovery checks for each metabolite to be analyzed. In conclusion, the method presented leads to less workload, more robustness, and a higher precision in metabolome analysis.

Carbon Isotopes↗

PubMed1998

[Effect of different prescriptions for tonifying kidney on RNA transcription activity in isolated nuclei and protein synthesis activity in free-cell extracts of the different organs of presenile mice].

OBJECTIVE: To study the anti-aging mechanism of Zuoguiyin (a prescription for tonifying Kindney Yin) and Yishenbao (a prescription for tonifying Kidney Yang) in presenile mice. METHODS: Using male, 14 months old presenile mice as a model of Kidney-Asthenia, the effects of Zuoguiyin and Yishenhao on RNA transcription activity in isolated nuclei and protein synthesis activity in free-cell extracts of the liver, brain, kidney and testes of the presenile mice were observed. RESULTS: RNA transcription activity in isolated nuclei and protein synthesis activity in free-cell extracts of the four organs of the presenile mice group were distinctly lower than those of young mice group (3 months old). After above-mentioned two prescriptions were given to the presenile mice for two months, RNA transcription activity in isolated nuclei and protein synthesis activity in free-cell extracts of the four organs of the two treated groups were obviously higher than those of control group (P < 0.05 or P < 0.01), but there was no difference between two treated groups. CONCLUSIONS: Both Zuoguiyin and Yishenbao could promote RNA transcription activity in isolated nuclei and protein synthesis activity in free-cell extracts of Kidney-Asthenia and presenile mice.

Aging↗

PubMed2003

Determination of pyridine and adenine nucleotide metabolites in Bacillus subtilis cell extract by sweeping borate complexation capillary electrophoresis.

With a growing interest in new areas of bioanalytical research such as metabolome analysis, the development of sensitive capillary electrophoresis (CE) methods to analyze sub-microM concentrations of analytes in biological samples is required. In this report, the application of CE with sweeping by borate complexation is used to analyze a group of seven pyridine and adenine nucleotide metabolites derived from bacteria Bacillus subtilis cell extracts. Nanomolar (nM) detectability of analytes by CE with UV photometric detection is achieved through effective focusing of large sample plug (approximately 10% of capillary length) using sweeping by borate complexation method, reflected by a limit of detections (S/N = 3) of about 2 x 10(-8) M. Changes in metabolites concentrations were observed in cell extracts when using either glucose or malate as the carbon source in the culture medium. Concentration of pyridine and adenine nucleotides in cell extracts varied widely from 78.6 (+/-7.6) microM for nicotinamide-adenine dinucleotide in malate to 0.66 (+/-0.12) microM for nicotinamide-adenine dinucleotide phosphate in glucose culture medium. Concentrations of metabolites in a single cell were also estimated at millimolar (mM) level. The method was validated in terms of linearity, sensitivity and reproducibility. The application of CE by sweeping borate complexation allows for sensitive and reproducible analyses of nucleotide metabolites in complex biological samples such as bacteria cell extracts.

Adenine Nucleotides↗

PubMed1986

The quantitative determination of 2'-deoxycytidine-5'-triphosphate in cell extracts by radioimmunoassay.

A radioimmunoassay (RIA) capable of quantitating dCTP in femtomolar amounts in cell extracts has been developed, and applied to human fibroblast cell lines and L5178Y mouse lymphoma lines. Cross reactivity of the antibody with CTP, though low (2.7%) has necessitated pre-RIA removal of CTP by either boronate affinity gel chromatography or sodium periodate oxidation. Fractions from the boronate gel column or aliquots of NaIO4-treated cell extract are quantitated directly by the RIA. Recovery of extracted dCTP standard taken through the entire procedure is quantitative and results are reproducible. Due to the high sensitivity of the quantitation step, dCTP can be accurately measured in relatively small numbers of cells--about 10(4) cells.

Animals↗

PubMed1991

DNA excision repair in mammalian cell extracts.

The many genetic complementation groups of DNA excision-repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell extracts and is defective in extracts from cell lines derived from individuals with the excision-repair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single-stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication.

Animals↗

PubMed1986

The mouse immunoglobulin heavy-chain gene enhancer contains sequences that inhibit transcription in vitro in HeLa cell extracts.

The effect of the cell-specific mouse immunoglobulin heavy-chain gene (IgH) enhancer on transcription from heterologous promoter elements was studied in vitro with a HeLa whole-cell extract. No stimulation of transcription could be seen under conditions in which an activation was observed with the simian virus 40 enhancer. We found, however, that a specific segment of the IgH enhancer region contains sequences which inhibit transcription in vitro.

Animals↗