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PubMed1995

The Ca(2+)-mobilizing actions of a Jurkat cell extract on mammalian cells and Xenopus laevis oocytes.

Randriamampita and Tsien (Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814) suggested that an acid-extracted fraction from a Jurkat cell line contains a messenger responsible for the coupling of calcium entry to the depletion of intracellular stores, i.e. capacitative calcium entry. We found that the extract, prepared as described by Randriamampita and Tsien, caused Ca2+ entry in 1321N1 astrocytoma cells which was not blocked by the D-myo-1,4,5-trisphosphate-receptor antagonist, heparin. In contrast to astrocytoma cells, when applied to mouse lacrimal acinar cells and rat hepatocytes the Jurkat extract always caused the release of intracellular Ca2+, followed by Ca2+ entry across the plasma membrane. This activity of the extract on lacrimal cells was blocked by either intracellular injection of heparin or extracellular atropine. Similarly prepared lacrimal cell extracts gave Ca2+ responses when applied to astrocytoma cells or lacrimal cells which were similar to those for Jurkat-derived extract. However, extracts from hepatocytes had no effect. In most Xenopus oocytes, the Jurkat extract had no effect, while in a few oocytes, the extract gave a [Ca2+]i response similar to that seen in lacrimal cells, that is, release of Ca2+ followed by Ca2+ entry. We conclude that the actions of the Jurkat cell extract are not consistent with its containing the long sought messenger for capacitative calcium entry. It is likely that this fraction contains a number of factors that mediate Ca2+ response in different cell types, possibly through receptor-mediated mechanisms.

Animals↗
PubMed1977

Search for inhibitors against herpes simplex virus type-I in cell extracts derived from human lymphoblastoid cell lines.

Cell extracts obtained from KB cells and 5 human lymphoblastoid cell lines including 2 from Burkitt's lymphoma (P3HR-1 and Raji), one each from nasopharyngeal carcinoma (no.223), acute lymphatic leukemia (MOLT-4) and a healthy person (NC-37) were tested for their inhibitory effects on the growth of herpes simplex virus type-1 (HSV-1) in green monkey kidney (GMK) cells by the plaque titration method. The relationship between the production of HSV-1 inhibitors and the degree of Epstein-Barr virus (EBV) genome repression in lymphoblastoid cells were also examined. Among the cell lines used P3HR-1 and no.223 cells produced a few EBV particles, Raji and NC-37 cells contained EBV genomes only, and MOLT-4 as well as KB cells were EBV genome-negative. The results revealed that P3HR-1 cell extract showed a tendency to inhibit HSV-1 growth in GMK cells but the other 4 lymphoblastoid cell lines and KB cells did not produce HSV-1 inhibitors, indicating that EBV genomes governing the formation of EBV structural antigens were not related to the production of HSV-1 growth inhibitors. The extracts from MOLT-4 cells, which are only a T lymphocyte cell line used in this study, stimulated HSV-1 growth in GMK cells significantly.

Antiviral Agents↗
PubMed1983

RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing.

Animals↗
PubMed2004

Preparation of Escherichia coli cell extract for highly productive cell-free protein expression.

As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.

Cell Fractionation↗
PubMed1995

Enhancement of in vitro transcription of human c-myc correlates with the binding of phosphorylated protein factors from HeLa cell extract.

Hela cell extract overphosphorylated with endogenous protein kinases was used for in vitro transcription of human c-myc DNA. The activation of transcription initiated from P1, P1a and P2 cap-sites was observed. Three phosphoproteins of 70, 35 and 23 kDa are found, whose binding to DNA correlates with activation of c-myc transcription. Sites of binding of these proteins to DNA are located.

Base Sequence↗
PubMed1989

Hydroxylation and Dechlorination of Tetrachlorohydroquinone by Rhodococcus sp. Strain CP-2 Cell Extracts.

A cell extract of a polychlorophenol-degrading bacterium, Rhodococcus sp. strain CP-2, isolated from chlorophenol-contaminated soil, was shown to dechlorinate tetrachlorohydroquinone, the first intermediate in pentachlorophenol and 2,3,5,6-tetrachlorophenol degradation. Degradation of tetrachlorohydroquinone was catalyzed by a soluble enzyme(s). The reaction sequence for complete dechlorination involved hydroxylation and three reductive dechlorinations, producing 1,2,4-trihydroxybenzene. All chlorines were thus removed from the polychlorinated compound before ring cleavage.

Journal Article↗
PubMed1986

Ataxia-telangiectasia cell extracts confer radioresistant DNA synthesis on control cells.

We have investigated in greater detail the radioresistant DNA synthesis universally observed in cells from patients with ataxia-telangiectasia (A-T). The approach employed in this study was to permeabilize cells with lysolecithin after gamma-irradiation and thus facilitate the introduction of cell extract into these cells. This permeabilization can be reversed by diluting the cells in growth medium. Cells treated in this way show the characteristic inhibition (control cells) or lack of it (A-T cells) after exposure to ionizing radiation. Introduction of A-T cells extracts into control cells prevented the radiation-induced inhibition of DNA synthesis normally observed in these cells. A-T cell extracts did not change the level of radioresistant DNA synthesis in A-T cells. Control cell extracts on the other hand did not influence the pattern of inhibition of DNA synthesis in either cell type. It seems likely that the agent involved is a protein because of its heat lability and sensitivity to trypsin digestion. It has a molecular weight (MW) in the range 20-30 000 D. The development of this assay system for a factor conferring radioresistant DNA synthesis on control cells provides a means of purifying this factor, and ultimately an approach to identifying the gene responsible.

Ataxia Telangiectasia↗
PubMed1976

Differentiation of lymphoid cells: the non-mitogenic induction of immunoglobulin production by thymus cell extract and thymus cell culture filtrate.

The cell-free medium in which thymocytes have been cultured (filtrate) as well as sonic lysates of thymocytes (extract) enhance immunoglobulin production when added to spleen cells during tissue culture. In spite of the requirement for foetal calf serum in the culture medium, production of the enhancing factor in thymocyte culture filtrates occurred even in the presence of a variety of metabolic inhibitors including NaN3, puromycin and hydroxyurea. Although DNA synthesis is required as a prelude to the induction of immunoglobulin production, two lines of evidence indicate that the enhancement produced in response to filtrate and extract occurs via a non-mitogenic process. First, neither cell-free agent was mitogenic toward spleen cells. Secondly, the enhancement of immunoglobulin production due to filtrate or extract was observed even in the presence of inhibitors of DNA synthesis. Multiple functions for thymocytes in the induction of immunoglobulin production are indicated by the findings that thymocytes restore immunoglobulin production of anti-thymocyte serum-treated spleen cells, whereas filtrate and extract, alone or in combination, do not have this capability. Furthermore, filtrate and extract failed to enhance the induction of DNP-group-specific antibody production by cells incubated with DNP-protein, but filtrate and extract could partially restore anti-DNP antibody production of such anti-thymocyte serum-treated cells. The role of thymocytes, filtrate and extract in the antigen-independent and the antigen-dependent induction of immunoglobulin production is discussed.

Animals↗
PubMed1993

[Comparative studies on concentration of berberine in plasma after oral administration of coptidis rhizoma extract, its cultured cells extract, and combined use of these extracts and glycyrrhizae radix extract in rats].

The present study was carried out to evaluate the bioequivalence between Coptidis Rhizoma and the cultured cells of Coptis japonica Makino var. dissecta Nakai to compare the concentration of berberine in the rat plasma after oral administration of both aqueous extracts. The concentration of berberine in the plasma after oral administration of both extracts was determined by HPLC. It was found that the values of time required for the maximum concentration (Tmax), the maximum concentration (Cmax) and the area under the plasma-time curve (AUC24h) of berberine in the rat plasma after oral administration of both extracts were about the same. It was also found that the values of Tmax, Cmax and AUC24h of berberine after oral administration of both extracts and an aqueous extract of Glycyrrhizae Radix were about the same as that of individual administration of both extracts. From these results, to evaluate the bioequivalence between Coptidis Rhizoma and cultured cells, it is important that the values of Tmax, Cmax and AUC24h of berberine after oral administration of both extracts are not different, and about the same values of berberine by the combined use of both extracts and the aqueous extract of other crude drugs are required.

Administration, Oral↗
PubMed1982

Specific protection of nucleotides in the lac operator from DMS methylation and DNase I nicking by crude bacterial cell extracts.

Crude bacterial cell extracts prepared from an Escherichia coli lacIq strain were shown to protect specific nucleotides in the lac operator from methylation by dimethyl sulfate (DMS) or digestion by DNase I, whereas no protection was observed using extracts prepared from a nearly isogenic lacI- strain. These experiments show that it is not necessary to use purified regulatory proteins in experiments designed to localize sequences on DNA which interact with proteins. Therefore, crude cell extracts should be useful in DNA "footprinting" experiments to define regions of DNA which bind to unknown regulatory proteins.

Base Sequence↗
PubMed1992

Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor. Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation.

A previous report from this laboratory (Reese, J.C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperature-sensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

Alanine↗
PubMed1983

Changes in actin-related gelation of crude cell extracts during differentiation of myeloid leukemia cells.

Gelation of extracts of a myeloid leukemia cell line (Ml) was compared before and after differentiation induced with conditioned medium (CM) from rat embryo cells. Although an extract of Mml cells, a macrophage line derived from Ml line, gelled when warmed in the presence of 2 mM MgCl2, undifferentiated Ml cells gelled only after dialysis and a supplement of exogenous actin. After differentiation had been induced, an addition of exogenous actin, but not dialysis, was needed for gelation. Small amounts of KCl always inhibited the gelation of the control Ml cell extracts, but they promoted gelation of the CM-treated Ml and Mml cell extracts. Thus, the dialysis required for gelation of the control Ml cell extract appears to be necessary for the exclusion of endogenous KCl. Several possible mechanisms for the KCl control of gelation, as well as different requirements of exogenous actin needed for gelation are discussed based on the results of our experiments.

Actins↗
PubMed1997

In vitro transcription of the Leptomonas seymouri SL RNA and U2 snRNA genes using homologous cell extracts.

A cell-free transcription system for the spliced leader (SL) RNA gene of the trypanosomatid Leptomonas seymouri has been developed. Accurately initiated transcription was achieved using cell extracts and a template in which the transcribed region of the SL RNA was replaced with a guanosine-less sequence (G-less cassette). The extract was also able to direct accurate initiation of RNA from an L. seymouri tagged U2 snRNA gene, which may be expressed via a transcriptional apparatus shared by the SL RNA gene. In vivo transcription analysis was used previously to define essential sequence components of the SL RNA gene promoter (Hartree D, Bellofatto V. Mol Biochem Parasitol 1995:71:27-39). A substitution mutation in the upstream promoter element (bp - 50 to - 70) markedly reduced transcription in vitro as did deletion of this and the middle promoter element (bp - 30 to - 40). Thus, the in vitro transcription system correctly responds to promoter mutations and is useful for investigating SL RNA and snRNA gene expression.

Animals↗
PubMed1971

Utilization of glucose by Clostridium thermocellum: presence of glucokinase and other glycolytic enzymes in cell extracts.

Clostridium thermocellum was shown to ferment glucose in a medium containing salts and 0.5% yeast extract. An active glucokinase was obtained with improved conditions for growth, assay, and preparation of cell extracts. Cell extracts appear to contain a glucokinase inhibitor that interferes with the assays at high protein concentrations. Glucokinase activity is stimulated about 60% by pretreatment with dithiothreitol. Little or no fructokinase or mannokinase activity was detected in cell extracts. The absence of glucokinase in mannitol-grown cells, the increase in glucokinase activity upon incubation of cell suspensions with glucose, and the lack of increase in activity when chloramphenical is added are evidence that glucokinase is an inducible enzyme. The following enzymes were detected in cell extracts (the enzyme activities are shown in parentheses are micromoles per minute per milligram or protein at 27 C): glucokinase (0.48), phosphoglucose isomerase (0.73), fructose 6-phosphate kinase (0.24), fructose diphosphate aldolase (0.59), glyceraldehyde 3-phosphate dehydrogenase (0.53), triose phosphate isomerase (0.13), phosphoglycerate kinase (0.20), phosphoglycerate mutase (0.20), enolase (0.28), pyruvic kinase (0.13), and lactic dehydrogenase (0.13). Glucose 6-phosphate dehydrogenase activity was absent or very low (0.0002) and 6-phosphogluconate dehydrogenase activity also was relatively low (0.015). From these data, it is proposed that carbohydrate metabolism in C. thermocellum proceeds by the Embden-Meyerhof pathway.

Alcohol Oxidoreductases↗