Effect of concanavalin a on ganglioside metabolism of human lymphocytes

The effect of Con-A on the incorporation of radioactivity from [¹⁴C]-glucosamine into gangliosides of human lymphocytes was investigated. Compared with non-stimulated lymphocytes there was increased incorporation into gangliosides and total lipids within the first 24 hours of exposure to Con-A. Ganglioside synthesis also occurred in later time intervals within the 96 hour incubation period. GM3 accounted for 80% of the labeled ganglioside in Con-A stimulated cells at all times studied. Thus ganglioside synthesis is not only associated with cellular division, but also occurs within a few hours of lymphocyte activation representing an extremely early prereplicative event.     

Specificity of Insertion by the Translocatable Tetracycline-Resistance Element Tn10

Genetic analysis of 131 independent transpositions of the tetracycline-resistance element Tn10 from a single site in phage P22 into the histidine operon of Salmonella typhimurium reveals that Tn10 insertions are not randomly distributed along this chromosomal target. The insertions occur in 22 different "clusters"; insertions within each cluster are very tightly linked in recombination tests. Tn10 insertions are not evenly distributed among the identified clusters. The existence of these clusters suggests that this chromosomal target contains particular genetic signals that guide Tn10 to particular preferred positions for insertion. Insertions within each cluster occur in both orientations with roughly equal frequency.--The relationship among different insertions within each cluster has been examined. The resolution of genetic mapping places an upper limit of about 50 basepairs on the distance between different insertions within a cluster. Different insertions within a cluster usually have the same reversion frequency; however, heterogeneity in reversion frequency has been detected in at least two clusters. For most clusters, the available data are consistent with the simple possibility that all insertions within a cluster are at identical positions; however, the data do not exclude other possibilities.     

Haemorrheological response to plasma exchange in Raynaud's syndrome.

Eight patients with Raynaud''s syndrome were treated by weekly plasma exchange for four weeks using a Haemonetics Model 30 Blood Processor. The mean whole-blood viscosity at a shear rate of 0.77/s was significantly lower after treatment, and the mean index of red-cell deformability was significantly improved. In four patients studied serially the mean percentage fall in whole-blood viscosity after a single plasma exchange was 49% at 0.77/s but only 14% at 91/s. All patients noticed symptomatic improvement including healing of ischaemic digital ulcers. In six patients the number of digital arterial segments containing detectable blood flow was measured by directional Doppler; in all six the number increased. It is concluded that plasma exchange is an effective means of haemorrheological treatment and may be beneficial in patients with digital ischaemia.     

Mutagenic effect on L5178Y mouse lymphoma cells by growth in ethylene oxide-sterilized polycarbonate flasks

Summary The mutation frequency of L5178Y mouse lymphoma cells to resistance to 5′-bromo-2′-deoxyuridine increased 6-to 14-fold after growth in ethylene oxide-sterilized polycarbonate culture flasks compared to growth in glass flasks. No comparable increase was observed when L5178Y cells were growth in identical polycarbonate culture flasks sterilized by autoclaving.     

Elementary steps of the (Na+ + K+)-ATPase mechanism, studied with formycin nucleotides.

1. Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a substrate for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), with properties similar to those of ATP. 2. FTP and formycin diphosphate (FDP) bind to the enzyme with high affinity and, on binding, the nucleotide fluorescence is enhanced 3-4-fold. It is therefore possible, with a stopped-flow fluorimeter, to measure the rates of binding and release of FTP and FDP under conditions in which turnover does not occur. 3. When the enzyme-FTP complex is exposed to conditions permitting turnover (Mg2+, Na+ +/- K+), changes in fluorescence occur which can be explained by supposing that they reflect the interconversion of states with or without bound nucleotides. A rapid fall in fluorescence, that we attribute to the rapid release of FDP from newly phosphorylated enzyme, is followed by a steady state in which low fluorescence suggests that little nucleotide is bound. Eventually, exhaustion of FTP allows rebinding of FDP to the enzyme, which is signalled by a rise in fluorescence. 4. The estimated rate of FDP release from newly formed phosphoenzyme is unaffected by the presence of K+ (0-2 mM) or the concentration of FTP (1-20 micron). 5. Experiments with [gamma-32P]FTP show that about 1 mol of 32P is incorporated per mol of enzyme. The rate of phosphorylation of the enzyme by [gamma-32P]FTP has been measured with a rapid-mixing-and-quenching apparatus. 6. Kinetic data from the fluorescence and phosphorylation experiments show that the behaviour of the enzyme, at least at the low nucleotide concentrations employed, is consistent with the Albers-Post model, and is difficult to reconcile with models in which K+ acts at or before the step in which FDP is released during turnover.     

The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.     

Transdetermination of Drosophila imaginal discs cultured in vitro

Imaginal discs of Drosophila melanogaster undergo transdetermination when cultured in vivo in the abdominal cavity of adult female hosts. We report here that leg discs cultured in vitro, in a recently developed system, also undergo transdetermination. Whether cultured in vivo or in vitro, leg discs produce a similar range of specific transdetermined structures. Moreover, in comparison to discs cultured in vivo, the discs cultured in vitro exhibit a similar correlation between the amount of growth and the total frequency of transdetermination.     

Protoplast induction inMicrasterias andCosmarium

Summary Protoplasts of the desmidsMicrasterias angulosa, M. denticulata, M. thomasiana andCosmarium turpinii were obtained by incubating cells in Waris' liquid medium + 0.3 M mannitol + 2% Cellulysin for 1–3 hours. One osmotically fragile protoplast was formed at the isthmus from the joint contents of both semicells. The resultant protoplasts were bright green and remained so for more than 5 days in the osmotically protective medium. The protoplast yield was better than 80%. The empty cell walls were not digested by the Cellulysin or by autolytic enzymes.     

The zinc content of yeast alcohol dehydrogenase

Analyses for zinc in high specific activity preparations of yeast alcohol dehydrogenase (YADH) indicate a metal content of 1.8–1.9 moles of zinc per mole of enzyme subunit. This zinc content is observed for YADH prepared from Bakers yeast by recrystallization from Am₂SO₄ containing 1 mM EDTA, followed by chromatography on DE-52 and Sephadex-G-200. YADH obtained from Boehringer-Mannheim is characterized by a variable specific activity: preparations with Sp. Ac. = 380–400 U/mg contain 1.8–1.9 moles of zinc per mole of subunit. Dialysis of YADH against EDTA (pH 8.5, 25°, under N₂) reduces the specific activity and zinc content in an approximately linear fashion down to a Sp. Ac. = 150 U/mg, consistent with the preferential loss of a single, weakly bound zinc per subunit which is essential for catalytic activity. Dialysis of YADH against 1 mM ZnCl₂ (pH 6.5–8.5, 25°, under N₂) does not lead to an increase in the zinc content of the enzyme, indicating that under these conditions zinc does not bind adventitiously to YADH. Dialysis against 50 mM CoSO₄ (pH 5.5, 25°, under N2, 60–90 hr) leads to an exchange of ≈ 40% of the enzyme-bound zinc by cobalt. Our preparations of YADH are consistently characterized by a zinc content of ≈ 2 per subunit and we are unable to reduce the zinc content of YADH by dialysis against EDTA without a concomitant loss in enzyme activity, in contrast to reports of one zinc per subunit [Veillon, C. and Sytkowski, A.J., BBRC $\text{67}$: 1499 (1975); Vallee, B.L. and Hoch, F.L., Proc. Nat. Acad. Sci. USA $\text{41}$: 327 (1955)]. The findings reported here, together with the observed structural similarities between YADH and horse liver alcohol dehydrogenase [Jornvall, H., Woenckhaus, C. and Johnscher, G., Eur. J. Biochem. $\text{53}$: 71 (1975)], suggest a role for zinc at both a structural and catalytic site in YADH.