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PubMed1976

The limiting effect of transfer RNA's on the rate of protein synthesis in cell extracts of rapidly growing tumors.

Using crude cell extracts from rapidly growing animal and human tumors, we found that (a) the addition of homologous transfer RNA (tRNA) to these extracts stimulated polypeptide synthesis two-to threefold, while addition of heterologous tRNA did not have a similar effect; (b) addition of homologous as well as heterologous ribosomal RNA was also stimulatory; and (c) both stimulatory effects were additive. The possibility that the effect of homologous tRNA could be mediated by contaminating material (such as the "translational control" RNA) seems to be rulted out by experiments with highly purified tRNA preparations, which did not contain even traces of 18 S, 7 S, 5 S, and smaller than 4S RNA's. Control experiments showed that no loss of tRNA occurred either during preparation of the cell extracts or under the conditions of in vitro protein synthesis. The results obtained suggest possible occurrence of a deficiency in specific isoaccepting tRNA's in rapidly growing solid tumors.

Animals↗

PubMed1992

Selective ability of S-phase cell extracts to dephosphorylate SV40 large T antigen in vitro.

Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein whose biological and biochemical functions appear to be modulated by phosphorylation. Recently, SV40 DNA replication in vitro has been shown to be activated by dephosphorylation involving the activity of a serine/threonine phosphoprotein phosphatase belonging to the type 2A class (PP2A) [Virshup, D.M., Kauffman, M.G. & Kelly, T.J. (1989) EMBO J., 8, 3891-3898]. To address the question of how specificity of PP2A activity towards T is regulated, an in vitro assay to study the process of T dephosphorylation was developed. Unlabeled extracts from cells enriched for various stages of the cell cycle were incubated with 32P-labeled, immunocomplexed T. Extracts from a population of cells enriched for S phase demonstrated a selective ability to dephosphorylate this labeled protein when compared with extracts prepared from G1- and M-phase cells. The time course of release from growth arrest demonstrated that this T-specific phosphatase activity occurred at the onset of host-cell DNA synthesis. In contrast, when using 32P-labeled phosphorylase a as the substrate, phosphatase activity appeared to be present throughout the cell cycle. The data presented here are consistent with the notion that PP2A activity towards T is regulated in a cell cycle-dependent manner.

Antigens, Viral, Tumor↗

PubMed1981

Immunobiological properties of 1-butanol-extracted cell surface antigens.

Extracts of viable 3-methylcholanthrene-induced murine sarcoma cells (MCA-F and MCA-2A) prepared using single-phase (2.5%) 1-butanol significantly retarded the outgrowth of the homotypic, but not the heterotypic, tumor of syngeneic C3H/HeJ mice. Butanol extracts specifically evoked a delayed hypersensitivity response in tumor-immune syngeneic mice, but not in alloimmune DBA/2J mice. Crude butanol extracts of MCA-F cells did not contain alloantigenic activity, as shown by their inability to block H-2 or Ia-specific antibodies in a complement-dependent cytotoxicity assay. Absorption of these same allospecific reagents with untreated or with butanol-extracted cells indicated that H-2 antigens remain associated with the cell surface during extraction. Thus, butanol appears to release tumor antigens, but not alloantigens, from the cell surface.

Animals↗

PubMed1995

Accurate in vitro transcription from circularized plasmid templates by plant whole cell extracts.

A convenient in vitro transcription system using monocot and dicot whole cell extracts and circular DNA templates has been developed. The system consists of incubating template and whole cell extract to generate initiation complexes, followed by addition of nucleotide triphosphates to support elongation, and primer extension assay to detect authentic transcripts. This in vitro transcription system required circularized templates and was essentially inactive with linearized templates. Accurate in vitro transcription of a rice phenylalanine ammonia-lyase (PAL) promoter-beta-glucuronidase (GUS) gene fusion and a tobacco sesquiterpene cyclase promoter-GUS gene fusion was examined in their homologous whole cell extracts, and the optimal concentrations for several reaction components, including DNA template, whole cell extract, monovalent and divalent cations, were determined for specific initiation from the in vivo start site. Transcription was inhibited by low concentrations of alpha-amanitin, demonstrating that the reaction was mediated by RNA polymerase II. Accurate transcription initiation was dependent on the TATA-box motif within the respective promoters. Based on the effect of delayed addition of sarkosyl at a concentration sufficient to inhibit transcription initiation but not elongation, three to four rounds of transcription were initiated in standard assays.

Base Sequence↗

PubMed1991

Phenotypic analysis of B-cells extracted from human periodontal disease tissue.

B-cells extracted from periodontal disease tissue were analyzed for the presence of activation markers using a range of monoclonal antibodies. In adult periodontitis (AP), 6% of B-cells expressed the IL-2 receptor (CD25) compared with 1-2% in peripheral blood and healthy or marginal gingivitis (H/MG) gingival B-cells. There was also an increase in the mean percentage of IgD-positive B-cells and a decrease in CD21 and CD22 expression. In both AP and H/MG lesions, 20-22% of the B-cells expressed CD23 compared with less than 5% in peripheral blood. As B-cells are activated by day 3 in culture and start differentiating into immunoglobulin-secreting cells by day 6, B-cell phenotypes were assayed at these times in this study. Following stimulation with the periodontopathic bacterium Porphyromonas gingivalis, the expression of CD23, CD21 and CD22 on B-cells extracted from AP lesions remained relatively constant over the 6-d culture period. However, with Fusobacterium nucleatum stimulation, there was a significant decrease in CD23, CD21 and CD22 expression after 3 d in culture, which corresponds to the activation time for B-cells. These results show that B-cells extracted from periodontal disease tissue display a range of activation markers and on stimulation, demonstrate differing responses to individual periodontopathic bacteria.

Adult↗

PubMed1992

An intrastrand d(GpG) platinum crosslink in duplex M13 DNA is refractory to repair by human cell extracts.

We have examined the ability of human cell extracts to repair the most frequent DNA adduct caused by the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II). A circular DNA duplex with an intrastrand d(GpG) crosslink positioned at a specific site was synthesized. Human cell extracts were unable to induce repair synthesis in a 29-base-pair region encompassing the adduct or in adjacent regions. The same extracts could repair a single defined 2-acetylaminofluorene lesion in a similar location. When molecules containing the platinum adduct were cleaved by Escherichia coli UvrABC enzyme, human cell extracts could perform repair synthesis at the damaged site, suggesting that human enzymes fail to make incisions near the d(GpG) crosslink but can complete repair once incisions are made. This result indicates that most repair synthesis in DNA damaged with multiple cis-diamminedichloroplatinum(II) adducts takes place at lesions other than the predominant d(GpG) crosslink. These data support the idea that the clinical effectiveness of cis-diamminedichloroplatinum(II) may be explained by the inefficient repair of the major DNA adduct caused by this drug.

Acetoxyacetylaminofluorene↗

PubMed1992

Calcium-sensitive, actin-related gelation of cell extracts of thermosensitive Chinese hamster lung cells capable of anchorage-independent growth.

The extent of actin-related gelation of extracts of thermosensitive Chinese hamster lung (CHL) cells capable of anchorage-independent growth was studied quantitatively by monitoring the total protein in the gel obtained by low-speed centrifugation. The gelation depended on the presence of ATP, KCl, MgCl2, and a reducing agent. Micromolar concentrations of Ca2+ and low doses of cytochalasin B inhibited the actin-related gelation of these extracts. The gelation was more sensitive to inhibition by Ca2+ and cytochalasin B when the extracts were prepared from cells cultured at the permissive rather than the nonpermissive temperature. When various ts mutants were examined, the half-maximal inhibitory dose (HMID) of Ca2+ and cytochalasin B for gelation of extracts of cells cultured at the nonpermissive temperature was between 1.25 and 2.19 times higher than that for extracts of the same cells cultured at the permissive temperature. The values of the HMID for Ca2+ and cytochalasin B changed shortly after the shift in temperature of cell cultures from the nonpermissive to permissive temperature. When cell extracts were incubated at the permissive temperature in vitro for only 15 minutes, these changes in values of HMID were also observed. Analysis of polypeptides of cell extracts and gel pellets on polyacrylamide gel electrophoresis suggested that the decrease in amount of a high-molecular-weight actin-binding protein (250 kDa) may play an essential role in the increased sensitivity to inhibition by Ca2+ and cytochalasin B of actin-related gelation in extracts of these ts mutants.

Actins↗

PubMed1984

Comparative toxicity, protective, histamine sensitizing and leucocytosis promoting activities in mice of Bordetella pertussis culture fluid, bacterial cells and cell extracts.

Bordetella pertussis bacterial cells, bacterial extracts, and concentrated culture supernatant fluid were comparatively examined for histamine sensitizing and leucocytosis promoting activities, toxicity (mouse weight gain test), immunoprotective potency and lipopolysaccharide bioassay. The activity of histamine sensitizing factor always paralleled that of leucocytosis promoting factor. In contrast, important differences were demonstrated regarding the toxicity and protective activity of the three preparations. Culture supernatant was more toxic and less protective than either bacterial cells or cell extract. Although the latter had lower protective potency than whole cells, its lower toxicity might lead to its consideration as a possible potential vaccine.

Animals↗

PubMed1977

The effects of red blood cell extracts on the proliferation of erythrocyte precursor cells, in vivo.

Saline incubation extracts of mature erythrocytes were assayed in vivo by a variety of techniques in order to study their ability to modify the proliferation of maturing erythroid cells. Using comparable extracts from granulocytes and lymphocytes, the specificity of the effect of the red cell extract for erythroid cells was confirmed by measurement of autoradiographic labelling indices, radio-iron incorporation and spleen colony growth. The erythroid cells were found to be very sensitive to the effects of the extract, as little as 10 microgram per mouse producing a maximum effect on iron incorporation. It was found that the extract does not block erythroid cells proliferation completely but simply lengthens the cell cycle, mainly by increasing the GI phase of the cycle. There was no effect on the committed erythroid precursor cells. The in vivo activity, specificity and non-toxicity to the cells, together with the cells' sensitivity to red cell extract suggest, therefore, that this inhibitor may play a physiological role in the control of red cell production.

Anemia↗

PubMed1977

Proteolytic activites in cell extracts of Trypanosoma cruzi.

Cell extracts of culture forms of Trypanosoma cruzi are capable of hydrolysing substances belonging to 4 different groups of protease substrates: (a) substrates for trypsin-like enzymes: benzoyl-arginine-p-nitroanilide and benzoylarginine-naphtylamide; (b) substrates for aminopeptidases: leucyl, lysl and glutamyl-beta-naphtylamide; (c) a substrate fochymotrypsin-like enzymes: carbobenzoxy-L-tyrosine-p-nitorphenylester, and (d) a nonspecific substrate for a broad range of proteases: azocasein. Some physico-chemical characteristics of each enzymic reaction were studied. They were found to be distint enought to allow attributing each hydrolytic activity to a separate enzyme.

Aminopeptidases↗

PubMed1984

Regulation and coupling of argECBH mRNA and enzyme synthesis in cell extracts of Escherichia coli.

Cell extracts from Escherichia coli were used to study both transcription and coupled translation of the argECBH gene cluster. Argininosuccinase (the argH enzyme) and N-acetylornithinase (the argE enzyme) were synthesized for 90 to 120 min, and hybridizable argECBH mRNA was synthesized for 60 min after the addition of a lambda or phi 80 dargECBH DNA template. L-Arginine (2.5 mM) repressed synthesis by argR+ extracts of argECBH mRNA 2-, to 3-fold, argE enzyme 5- to 8-fold, and argH enzyme 20- to 60-fold. Repression was specific for L-arginine, and argR extracts were insensitive to added L-arginine. The argECBH mRNA made under conditions of restricted protein synthesis had reduced ability to function in the formation of the argE and argH enzymes and was found to be predominantly 6 to 8S in sucrose density gradients. When protein synthesis was allowed, the mRNA formed was functional, and large amounts of 14 to 23S argECBH mRNA appeared on sucrose gradients. An S-100 supernatant freed of ribosomes was capable of producing hybridizable arg mRNA, but significant functional message was only produced when ribosomes were present. When purified RNA polymerase was used, the formation of short 6 to 8S argECBH mRNA was dependent upon added rho protein. The data suggest that rho-dependent sites in the argECBH operon allow early termination of mRNA synthesis when transcription is not coupled to active enzyme synthesis.

Amidohydrolases↗

PubMed1990

Variations in Feulgen stainability of epithelial parenchymal cells extracted from paraffin-embedded salivary gland specimens.

The variations of Feulgen stainability of cells extracted from paraffin-embedded archival specimens for DNA assessment by means of image cytometry (ICM) were investigated in normal salivary gland parenchyma. The Feulgen stainability of the deparaffinized, rehydrated, and disaggregated preparations was found to exhibit variations of up to 300%, expressed by the mean of integrated optical density (IOD), when a routine procedure was applied to a first series of Cytospin preparations of disaggregated specimens. When measured in nondisaggregated tissue sections, only negligible variations were observed. After minimization of the mechanical strains to the cellular material in the Cytospin preparations in a second series, the variations in Feulgen stainability were found to be considerably lower. The findings indicate that the main reason for variations in the Feulgen stainability of extracted cells is, most likely, the disaggregation procedure itself. Factors such as initial treatment of the specimens, duration and kind of formalin fixation, and length of storage time periods seem to be of minor importance. Retrospective studies on paraffin-embedded specimens require a carefully controlled tissue type-adapted disaggregation procedure. In addition, we concluded that the interpretation of histograms, obtained by means of ICM DNA assessments in Cytospin preparations of archival material, requires a well-defined internal specific standard.

Coloring Agents↗

PubMed2003

Biotransformation of halophenols using crude cell extracts of Pseudomonas putida F6.

Crude cell extracts of Pseudomonas putida F6 transformed 4-substituted fluoro-, chloro-, bromo- and iodo-phenol without the exogenous addition of cofactors. The rate of substrate consumption decreased with increasing substituent size (F>Cl>Br>I). Biotransformations resulted in greater than 95% utilisation of the halogenated substrate. Product accumulation was observed in incubations with 4-chloro, 4-bromo- and 4-iodo-phenol. These products were identified as the corresponding 4-substituted catechols. Transformation of 4-fluorophenol did not result in the accumulation of the corresponding catechol; however, manipulation of the reaction conditions by incorporation of ascorbic acid culminated in the formation of 4-fluorocatechol. Cell extracts of P. putida F6 also showed activity towards a 3-substituted phenol, namely 3-fluorophenol, resulting in the formation of a single product, 4-fluorocatechol.

Biotransformation↗

PubMed2000

Activation of fibroblasts in collagen lattices by mast cell extract: a model of fibrosis.

BACKGROUND: Mast cells are resident connective tissue cells able to secrete numerous inflammatory mediators in response to tissue aggression and might be implicated in the fibrotic processes. OBJECTIVES: To study the effects of mast cell products on fibroblast activity in connective tissues. METHODS: Mast cell extract was prepared by sonication of pure mast cell preparations obtained by peritoneal lavage of rats and added to the culture medium of fibroblast-populated collagen lattices. RESULTS: Mast cell extract was able to decrease the contraction of the collagen lattices, to stimulate total protein and collagen synthesis, and to increase the expression and activation of gelatinase A/MMP-2. CONCLUSION: These data are consistent with the hypothesis that mast cells in connective tissue may be responsible for fibroblast activation at the early phases of tissue repair and fibrosis.

Adult↗

PubMed1981

Transcription of Simian virus 40 DNA in a HeLa whole cell extract.

Extracts of HeLa cells containing RNA polymerase II and other factors recognize specific sites on linear simian virus 40 (SV40) DNA for initiation of transcription. The most prominent RNA products transcribed from the early region of SV40 and th E strand are initiated at sites 0.67 and 0.655 on the SV40 map. These two RNAs are synthesized by polymerase II. Their 5' termini were positioned by sizing transcripts that extend from the initiation site to the end of the template restriction endonuclease fragment, and by S1-nuclease mapping of unlabeled RNA using DNA probes labeled at their 5' termini. The limit of resolution of mapping of 5' termini is approximately 25 nucleotides. RNAs with 5' termini at similar positions have been found during characterization of mRNAs produced in infected cells. Thus, the whole cell extract is probably initiating transcription on linear SV40 DNA in vitro at the same sites as RNAs synthesized in vivo. Two other processes frequently involved in mammalian cell mRNA biosynthesis, creation of specific 3'-terminal polyadenine tracts and RNA splicing, were not detected during the course of these studies.

Cell Transformation, Viral↗

PubMed1997

Inhibition of DNA synthesis and G1/S-phase transition in normal human fibroblasts elicited by a heat-labile trans-acting factor in gamma-irradiated HeLa cell extracts.

Proliferating human cells exposed to ionizing radiation show complex cellular responses including a delay in progression through various phases in the cell cycle. These cell cycle checkpoints are regulated by mitogenic signaling pathways which transduce the extracellular signals to the cell cycle control machinery. In this study we demonstrate that microinjection of a cellular extract, prepared from gamma-irradiated (40 Gy) HeLa cells, into the cytoplasm of normal human fibroblasts results in suppression of DNA replicative synthesis, indicating the presence of a trans-acting DNA synthesis-inhibiting factor(s). The addition of this same extract to the culture medium for a short time (< or = 2 h) also inhibits DNA synthesis in human fibroblasts, affecting both replicon initiation and DNA chain elongation processes. Moreover, a 2-h incubation of the fibroblast cultures with the extract causes a transient delay in cell progression from G1 to S phase coupled with up-regulation of the p53 tumor suppressor protein. Both the DNA synthesis-inhibiting and G1-phase-blocking activities are reduced markedly when the extract is heated (80 degrees C; 10 min) prior to its addition to the culture medium. On the other hand, pretreatment of the fibroblast cultures with KN62, an inhibitor of calmodulin-dependent kinase II (CaMKII), serves to abrogate the inhibitory effect of the extract on DNA synthesis without influencing its ability to induce the G1-phase block. These results are compatible with the presence in HeLa cell extracts of a heat-labile trans-acting factor that triggers, in normal human cells, the activation of (1) a CaMKII-dependent signal transduction pathway mediating suppression of DNA synthesis and (2) a p53-dependent pathway mediating G1-phase checkpoint control.

Cell Cycle↗

PubMed1984

An improved method for the quantitative determination of deoxyribonucleoside triphosphates in cell extracts.

The analysis of deoxyribonucleoside 5'-triphosphates (dNTPs) in cell extracts by high-pressure liquid chromatography [C. Garrett , and D.V. Santi (1979) Anal. Biochem. 99, 268-273] requires the prior, selective degradation of ribonucleoside 5'-triphosphates ( rNTPs ) that are present in the extracts in large quantities. When this method was used for quantifying the dNTPs in mammalian cell extracts, the presence of an interfering peak in the HPLC between the peaks for dTTP and dATP was observed. This unwanted peak sometimes overlapped with that of dATP, depending on the pH of the eluant. It was found that the material which gave this peak was formed during the periodate oxidation of rNTPs in the presence of methylamine, and that it could be removed by changing the order of addition of the reagents in the procedure, i.e., the methylamine was added only after the excess periodate was decomposed, instead of adding it together with periodate, as given in the original procedure. Furthermore, an addition of deoxyguanosine to the reaction mixture was found to be effective in preventing the partial loss of dGTP in the oxidation procedure. By using this improved method, the dNTP contents of the extracts of Ehrlich ascites tumor cells have been measured in an accurate and reproducible manner. The analysis requires about 10(6) cells, and all four dNTPs can be quantified in 2.5 h, starting from the harvest of the cells.

Animals↗

PubMed1983

Assay of 2',5'-oligoadenylate phosphodiesterase activity in mouse L-cell extracts.

A rapid and convenient assay for adenylyl(2' leads to 5')adenosine(A2'p5'A) or adenylyl(3' leads to 5')adenosine(A3'p5'A) phosphodiesterase activities is described. The dinucleotides A3'p5'A and A2'p5'A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [3H]A3'p5'A or [3H]A2'p5'A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2'p5'A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2'p5'A as extracts from interferon pretreated cells.

Adenosine Monophosphate↗