Altered thymidine kinase or thymidylate synthetase activities in 5-fluoro deoxyuridine resistant variants of mouse neuroblastoma.

Abstract: Abstract-We have previously described a 5-fluorodeox yuridine (FUdR) resistant neuroblastoma variant, possessing normal levels of ATP: thymidine-5-phosphotransferase (EC 2.7.1.21) [trivial name: thymidine kinase (TK)] but an 8-fold elevation in methy1enetetrahydrofolate:dUrd-5′P C-methyltransferase (EC 2.l.l.b) [trivial name: thymidylate synthetase (TS)] relative to the drug-sensitive parental clone. This variant possesses elevated levels of the parental TS species, 30% of which is uninhibitable by in vivo pulses of FUdR, suggesting the subcellular compartmentalization of this enzyme. We contrast this variant with a second FUdR resistant clone isolated from an ethyl-methane-sulfonate mutagenized population of the parental clone. This variant displays a 96% reduction in TK specific activity, despite normal FUdR and thymidine uptake rates, demonstrating the independence of thymidine phosphorylation and uptake. Grown without drug, its resistance declines (half-life of 15 cell divisions) with its TK specific activity rising to a plateau of 16% of the parental level after 56 cell divisions. Thymidine (1.0μM) protects the TK+ but not the TK- variants from FUdR induced growth inhibition but is without effect on TS specific activity. Unlike Tetrahymena (DICKENS et al., 1975), neuroblastoma TS activities appear not to be regulated by adenosine or guanosine cyclic nucleotide levels.     

Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase)

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the sialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens$\text{N-}\text{acetylneuraminate}$ glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspension medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca²⁺ in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that a restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.     

The Role of Estrogens as a Risk Factor for Stroke in Postmenopausal Women

The estrogen component in oral contraceptives is generally thought to increase the risk of stroke in users as compared with controls. If this were true, an increased risk for stroke also should be seen among postmenopausal women using estrogens as compared with nonusers. To test this hypothesis, the charts of 198 postmenopausal patients who had had strokes were compared with those of 396 controls for estrogen use and for the associated risk factors of diabetes, hypertension and coronary artery disease. The difference between estrogen use in the study population versus controls proved not significant, and the use of estrogens did not significantly influence the distribution of the above risk factors. We concluded that the use of estrogens in physiological replacement doses does not increase the risk of stroke in postmenopausal women.     

Fermentation of pentose sugars to ethanol and other neutral products by microorganisms

The current and projected shortage of petroleum and natural gas has led to renewed interest in processes for the microbial conversion of renewable biomass resources to liquid and gaseous fuels. Pentose sugars represent a significant fraction of the total fermentable carbohydrate content of biomass. A number of biochemical pathways are known for the conversion of pentose to ethanol and other neutral products. Beside ethanol, potential neutral fermentation products include 2,3-butanediol, acetone, isopropanol, butanol and hydrogen. Other products include carbon dioxide and organic acids. Specific ethanol-producing fermentations are reviewed, and future directions for research and development are suggested.     

Purification and properties of guinea pig antithrombin III

Guinea pig antithrombin III has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography. The final product was homogeneous as judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of antithrombin III was determined by progressive inactivation of thrombin coagulant and amidolytic activity. Heparin cofactor activity was demonstrated by immediate inactivation of thrombin by antithrombin III in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin III occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities of heparin.     

Hydrogen exchange kinetics of human hemoglobins. The pH dependence of solvent accessibility in cyanomet-, oxy-, and deoxyhemoglobin.

The hydrogen exchange kinetics of human oxy-, deoxy-, and cyanomethemoglobin have been measured as a function of pH by the tritium tracer method. At 5 degrees C and in phosphate buffer both liganded and unliganded forms of ferrohemoglobin exhibit deviations from the regular pH dependence of exchange that is characteristic of cyanomethemoglobin. In oxyhemoglobin, the deviation from the normal exchange pattern is centered at pH 7.4 and is in the direction of increased exchange or solvent accessibility. The effect in deoxyhemogloin, while occurring at the same pH and being of the same order of magnitude, is in the opposite direction, thus suggesting a pH-induced conformational transition leading to a less accessible structure. The width of these pH-induced deviations in solvent accessibility is approximately 1 pH unit in both cases. We propose a model in which specific interactions between charged groups in both froms of ferrohemoglobin account for these deviations.     

Immunity to Plasmodium berghei yoelii in mice. II. Specific and nonspecific cellular and humoral responses during the course of infection.

The kinetics of various specific and nonspecific immunologic responses were examined in BALB/c mice infected with 17X nonlethal Plasmodium berghei yoelii (a self-limiting infection). The sequence of events after infection was characterized by rapid sensitization of splenic T cells to malaria antigen and polyclonal B cell activation, followed by a period of depressed splenic proliferative responses in vitro to mitogens (PHA and LPS) and malaria (specific) antigen. At the same time, suppressed primary in vitro splenic PFC responses to trinitrophenyl-aminoethylcarbamylmethyl-Ficoll (TNP-F) were seen. This suppression was an active process requiring adherent cells. During this period, levels of antimalarial antibody also increased exponentially. As the infection was cleared, splenic malaria antigen-specific proliferative responses were again observed and splenic PFC and in vitro mitogen responses returned to preinfection levels after variable periods of time. Both splenic proliferative responses to malaria antigen and antimalarial antibody responses remained persistently elevated. In addition, some responses were examined in mice infected with 17X lethal P.b. yoelii (a fatal infection); in comparison to the early responses of mice infected with the nonlethal substrain, there was a decrease and delay in the development of a splenic T cell response to malaria antigen and a blunted antimalarial antibody response.     

Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes

For very narrow channels in which ions and water cannot overtake one another (single-file transport), electrokinetic measurements provide information about the number of water molecules within a channel. Gramicidin A is believed to form such narrow channels in lipid bilayer membranes. In 0.01 and 0.1 M solutions of CsCl, KCL, and NaCl, streaming potentials of 3.0 mV per osmolal osmotic pressure difference (created by urea, glycerol, or glucose) appear across gramicidin A-treated membranes. This implies that there are six to seven water molecules within a gramicidin channel. Electroosmotic experiments, in which the water flux assoicated with current flow across gramicidin-treated membranes is measured, corroborate this result. In 1 M salt solutions, streaming potentials are 2.35 mV per osmolal osmotic pressure difference instead of 3.0 mV. The smaller value may indicate multiple ion occupancy of the gramicidin channel at high salt concentrations. Apparent deviations from ideal cationic selectivity observed while attempting to measure single-salt dilution potentials across gramicidin-treated membranes result from streaming potential effects.     

In vitro growth of cytotoxic human lymphocytes. I. Growth of cells sensitized in vitro to alloantigens.

Human lymphocytes sensitized to allogeneic determinants in vitro were continuously cultured on medium prepared from phytohemagglutinin-(PHA-P) stimulated human mixed lymphocyte cultures. With such human-conditioned medium (HCM), human peripheral lymphoid cells could be grown in culture for over 90 days with a doulbing time of about 54 hr. Cytotoxic lymphocytes could be grown in culture for this time with no loss of cytotoxicity or change in cytotoxic specificity.