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At least 19 recordsLinked to original sources

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↗

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↗

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↗

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↗

PubMed1975

Interferon treatment of Ehrlich ascites tumor cells: effects on exogenous mRNA translation and tRNA inactivation in the cell extract.

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S60INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30INT between tRNA from interferon-treated cells and tRNA from control cells. Futhermore, no difference was found in the rate of inactivation in S30INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under out conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation fo exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.

Animals↗

PubMed1979

Characterization of a C21 neutral steroid hormone transforming enzyme, 21-dehydroxylase, in crude cell extracts of Eubacterium lentum.

A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.

Autoradiography↗

PubMed1977

Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts.

The gelation induced by warming (to 25 degrees C) the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 was studied in the presence of myosin and heavy meromyosin (HMM). Myosin mixed with extract induces shrinkage of the gel, but jelled extract or myosin alone does not shrink. In the concentration range, 0.14-1.04 mg/ml of myosin, the degree of shrinkage is roughly proportional to the concentration of myosin. Supplementa MgCl2 also promotes shrinkage. HMM (0.4-0.8 mg/ml) can inhibit gel formation by extract in tubes or floated on a sucrose cushion. Gel electrophoresis of gels shrunken by added myosin or electrophoresis of the proteins which can be sedimented from extract after incubation in the presence of HMM indicate that both myosin and HMM interfere with the changes in sedimentability of the high molecular weight protein (HMWP) thought to participate (together with actin) in gel formation in HeLa cell extracts (R. R. Weihing, 1976. J. Cell Biol. 71:303-307). These results, together with previous results showing that actin is present and that HMWP is enriched in the plasma membrane fraction of HeLa cells (R. R. Weihing, 1976. Cold Spring Harbor Conf. Cell Proliferation. 3:671-684), point to the possibility of dynamic changes in the interactions of HMWP or myosin with actin in processes of movement occurring at the cell surface.

Actins↗

PubMed1975

Glucanases in Schizosaccharomyces. Isolation and properties of an exo-beta-glucanase from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis.

(11 Cell extracts and extracellular culture fluids of species of the yeast genus Schizosaccharomyces exhibited exo-beta-(1 leads to 3)- and exo-beta-(1 leads to 6)-glucanase (EC 3.2.1.-) activities. (2) Using a combination of Sephadex G-100 and DEAE-cellulose chromatography, the exo-beta-(1 leads to 3)-glucanases from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis were purified extensively. The enzymes from either location exhibited similar purification and other properties. (3) The purified enzymes hydrolysed the beta-(1 leads to 6)-glucosidic linkage in addition to the beta-(1 leads to 3) linkage. Heat denaturation, inhibition and electrophoretic studies indicated that both hydrolytic activities were properties of a single protein. Laminarin and pustulan hydrolysis followed Michaelis-Menten kinetics. The Km and V for laminarin hydrolysis were 6.25 mg/ml and 350 mumol of glucose released/min/mg protein, and for pustulan they were 166 mg/ml and 52 mumol of glucose released/min/mg protein. (4) The exo-beta-glucanase was assigned a molecular weight of 43 000. (5) the purified enzyme failed to hydrolyse isolated cell walls from either baker's yeast or Schizosaccharomyces pombe or to induce protoplast formation from intact cells of S. japonicus var. versatilis or Saccharomyces cerevisiae.

Ascomycota↗

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↗

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↗

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↗

PubMed1976

Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA.

The translation of encephalomyocarditis virion RNA in extracts from interferon-treated L-cells is inhibited by the addition of double-stranded RNA (dsRNA) at 400 ng/ml. A similar inhibition in response to dsRNA is seen in control cell extracts supplemented with small amounts of a postribosomal supernatant fraction from interferon-treated cells (interferon cell sap): Neither interferon cell sap nor dsRNA alone is inhibitory in control systems. The inhibition is much reduced if translation is carried out at low ATP concentrations.Conversely, the inhibitory capacity of the interferon cell sap is increased 100-fold if it is preincubated with dsRNA and ATP prior to its addition to the protein-synthesizing system. After this preincubation all detectable dsRNA can be removed without any diminution of the inhibitory activity of the cell sap. These results are compatible with a two-step model for the inhibition in which a pre-inhibitor is activated by dsRNA, the activated inhibitor then interacting with the protein synthesis system to inhibit translation.

Adenosine Triphosphate↗

PubMed1977

Competence of soluble cell extracts as microtubule assembly systems. Comparison of simian virus 40 transformed and nontransformed mouse 3T3 fibroblasts.

Soluble cell extracts from simian virus 40 transformed mouse 3T3 fibroblast cells (SV101) and nontransformed mouse BALB/c-3T3 cells were compared as microtubule assembly systems. Extracts from the transformant were found to be at least as competent for microtubule assembly under standard conditions as those from normal cells. This observation proves that the defects in cytoplasmic microtubule skeletons reported in the literature for various transformed cell lines are not due to the loss of integrity of the tubulin molecule itself, but rather to transformation-dependent changes in the regulatory mechanisms controlling in vivo microtubule assembly.

Cell Line↗

PubMed1976

Comparison of antigens in sonic and pressure cell extracts of Mycobacterium tuberculosis.

Comparisons were made of the yield, chemical content, and biological activity of filtrates and extracts obtained by sonic and pressure cell disruption of bacilli from 4- and 8-week-old Proskauer and Beck cultures of the H37Rv strain (TMC no. 102) of Mycobacterium tuberculosis. The culture filtrates were dialyzed, freeze-dried, reconstituted in saline, and sterilized by membrane filtration. The viable bacilli were washed and resuspended in distilled water and subsequently disrupted either by sonication in the cold for 15 or 30 min or by treatment at 20,000 or 40,000 lb/in2 in a pressure cell. The resulting extracts were clarified by centrifugation, concentrated, and sterilized by filtration. All preparations were adjusted to contain 10 mg of solids (dry weight)/ml and were analyzed quantitatively for protein, deoxyribonucleic acid, ribonucleic acid, polysaccharide, and lipid content. Separation patterns obtained by gradient acrylamide gel electrophoresis, as well as by one- and two-dimensional immunoelectrophoresis, provided the basis for qualitative comparisons of the culture filtrates and cell extracts. Three-point dose-response curves also were used to compare the preparations for skin test reactivity in BCG-vaccinated guinea pigs. It was concluded that, although there were no consistent differences in chemical content or biological activity between the preparations, a 15-min sonic treatment appeared to be the most suitable method for preparation of bacillary extracts based on yield of active components and ease of preparation.

Animals↗

PubMed1978

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. III. The determination of 14C-labeled 6-thiopurines in L5178Y cell extracts using high-pressure liquid cation-exchange chromatography.

A method is presented for the separation of 6-thiopurine bases and ribonucleosides, of sulphate anions and of common purine bases and oxidized purines by means of high-pressure liquid cation-exchange chromatography using a 0.18 X 100 cm column, filled with Beckman M71 resin, and eluted with 0.4M ammonium formate, pH 4.6, at a linear flow velocity of 5.2 cm/min at 50 degrees C. The method has been applied to the separation and quantitative determination of 14C-labeled 6-mercaptopurine metabolites in HClO4 extracts of L5178Y murine lymphoma cells. Distribution patterns of 14C radioactivity within the cells after a 24 h incubation period with (8-14C)-labeled 6-mercaptopurine have been established. The indentification of 6-mercaptopurine metabolites, such as 6-thioxanthosine ribonucleotide, 6-thioinosinic acid, 6-thioguanylic acid, 6-methylthioinosinic acid, and 6-thiouric acid, after the digestion of the extracts with alkaline phosphatase has been confirmed using the behaviour of each compound in enzymatic peak-shifting analyses with purine nucleoside phosphorylase and the corresponding elution volumes of 6-thiopurine bases and ribonucleosides as proofs. According to the specific radioactivity of the (8-14C)-labeled 6-mercaptopurine batch, the amounts of the various 6-mercaptopurine metabolites in about 6% of the total HClO4 extract of 1.6 . 10(8) labeled cells have quantitatively been determined as 1--130 pmol. The intracellular concentration of 6-thiopurines was determined at 1.4 . 10(-5)mol/1.

Alkaline Phosphatase↗

PubMed1976

Cytochalasin B inhibits actin-related gelation of HeLa cell extracts.

When the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 is incubated at 25 degrees C, it gels, and actin and a HMWP are progressively enriched in the extract and in gel isolated from extract. CB (greater than or equal to 0.25 muM) inhibits gelation and specifically lowers the concentrations of actin and the HMWP in the fraction which sediments at 100,000 g after incubation. These results indicate that actin and HMWP are partly disaggregated by cytochalasin treatment, and thus that their aggregation is related gelation. Inasmuch as previous results showed that actin is present and HMWP is enriched in the plasma membrane fraction of HeLa cells, the results also point to a possible relation between plasma membrane-associated gel and in vivo effects of CB.

Actins↗

PubMed1975

Removal of unwanted proteins from cell extracts by means of antiserum.

The efficiency of removal of soluble proteins in cell-free bacterial extracts by means of antiserum from rabbits immunized with similar extracts was measured. Precipitation followed by Sephadex gel-chromatography was used. Up to 80% (exceptionally 90%) removal could be obtained. The method might be applied to enrichment for "foreign" cell-extract components, for example, viral products in virus infected cells. Tests for the specificity of the method are also presented.

Animals↗