Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 μg/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl- -glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl- -glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4−7 × 10⁻⁸/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.     

Mapping and isolation of large peptide fragments from bovine neurophysins and biosynthetic neurophysin-containing species by high-performance liquid chromatography

A peptide mapping procedure suitable for rapid analysis and peptide recovery was devised for the neurophysins. Tryptic fragments of performic acid-oxidized bovine neurophysins I and II were fractionated by reverse-phase high-performance liquid chromatography using γ-cyanopropyl-bonded columns. Elutions were achieved using a gradient from triethylammonium phosphate buffer to mixtures of this buffer with increasing proportions of acetonitrile. All tryptic fragments, except for dipeptides, were separated. Assignments of eluted peaks to particular authentic neurophysin peptides were achieved by collection of peaks and determination of their amino acid compositions. The use of this peptide mapping procedure to detect subpicomole amounts of neurophysin peptides in cell-free biosynthetic products was demonstrated.     

Potentiation of the in vitro cytotoxic response to syngeneic lymphoma cells by soluble products of tuberculin-sensitive lymphoid cells stimulated with PPD

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus induced lymphoma have previously been shown to differentiate into cytotoxic effector cells following restimulation with tumor cells in vitro. Previous work has also demonstrated that the addition of PPD-primed syngeneic spleen cells and PPD to cultures of (C58NT)D-primed spleen cells will potentiate the in vitro cytotoxic response to tumor antigens. In the studies presented here, the potentiating effect was found to be mediated by a soluble factor(s) released by nonadherent cells from BCG-primed rats. The release of this immunopotentiating factor(IPF) required the presence of PPD and varied with the concentration of PPD added. IPF was produced by BCG-primed spleen, lymph node, and thymus cells. Maximal production of IPF in PPD-stimulated cultures was obtained after 6–12 hr of incubation. Supernatants obtained after 30 hr of incubation lacked apparent IPF activity when tested initially, but activity was recovered after mild heat treatment. Recovery of IPF activity after heat exposure is best explained by the presence of a heat-labile inhibitor. IPF itself is stable to heat treatment to 56 °C for 40 min. IPF was also shown to be capable of enhancing immune responses to histocompatibility antigens in vitro.     

Effects of tetracyclines on aldosterone- and insulin-mediated Na+ transport in the toad urinary bladder

The effect of oxytetracycline and demethylchlortetracycline on aldosterone- and insulin-mediated Na⁺ transport (short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. Oxytetracycline had little or no effect on either basal or aldosterone-mediated Na⁺ transport. In contrast, demethylchlortetracycline markedly inhibited both basal and aldosterone-mediated Na⁺ transport. Furthermore, demethylchlortetracycline inhibited the aldosterone response significantly out of proportion to its effects on basal Na⁺ transport. Neither of the drugs had an effect on insulin-mediated Na⁺ transport. Consequently, the natriuresis observed in certain patients treated with demethylchlortetracyline may be related to drug-induced renal resistance to the effects of aldosterone.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase.

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade-G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade- mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade-G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade-G is defective in phosphoribosylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.     

Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man

Levels of norepinephrine (NE) in human plasma have been determined by a radioenzymatic technique sufficiently sensitive to measure 0.014 ng NE per ml plasma. Several procedures which raise plasma NE levels have been compared and a standard procedure developed to evaluate sympathetic neuronal function based on the increments in plasma NE produced by postural change and a standard amount of exertion. The mean basal level of NE in plasma of 74 resting, supine, normal subjects ranging in age from 10 to 70 (mean 32.7 years) was 0.292 ± 0.016 (± SEM) ng/ml and ranged from 0.112 to 0.738 ng/ml. There was a significant correlation between age and basal levels of NE (L.R. = 0.33, p < 0.01). In 44 subjects who stood for 5 minutes after the basal sample of blood was obtained, the mean plasma level of NE increased to 0.538 ± 0.044 ng/ml and further increased to 0.778 ± 0.080 ng/ml after a subsequent isometric hand grip for 5 minutes.     

Interpersonal relationships and personal space: Research review and theoretical model

This article reviews research concerning interpersonal distance as a function of interpersonal relationships, attraction, and reactions to spatial invasion. To integrate research findings, we propose a simple model, based on the idea that people seek an optimal distance from others that becomes smaller with friends and larger for individuals who do not expect to interact. The model describes comfort-discomfort as a function of interaction distance in three situations: interacting friends, interacting strangers, and strangers who do not expect interaction. These three personal space profiles are discussed in terms of qualifying variables, such as seated vs. standing interaction, sex composition of the dyad, intimacy of conversation topics, and situational variables.A version of the theoretical section of this article was presented at the 82nd annual convention of the American Psychological Association in a symposium entitled Getting Close: Personal Space and Privacy, New Orleans, 1974. An earlier version of the material appears in a book by Irwin Altman (1975).     

Monamine oxidase in outer membrane of skeletal muscle mitochondria.

(1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay procedure for the assay of monoamine oxidase in muscle mitochondria is described. It is based on teh procedure using side-chain [2-14C]-tryptamine as substate described by Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate concentration of 0.25 mM. (3) The Km of the muscle mitochondrial enzyme at pH 8.0 is 1.34 - 10(-5) M and that of the liver enzyme under the same conditions is 2.5 - 10(-5) M. Muscle mitochondria contain only one quarter of the activity of enzyme present in liver mitochondria. (4) Monoamine oxidase is shown to be in the outer membrane of skeletal muscle mitochondria and thus to be a suitable marker enzyme for use in the fractionation of these mitochondria.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.