Plasmalemma Chloride Transport in Chara corallina: Inhibition by 4,4'-Diisothiocyano-2,2'-Disulfonic Acid Stilbene

Chloride transport, presumably via a Cl⁻-2H⁺ co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter² per second) was stimulated 4-fold by an 18-hour Cl⁻ starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter² per second after a 60-minute pre-exposure to 0.5 millimolar 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter² per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl⁻ influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl⁻ to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl⁻ restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl⁻ influx by inhibiting the putative plasmalemma H⁺-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity.

The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl⁻, in contrast to the presence of Cl⁻ during pretreatment of controls. The differential effect could result from competition between Cl⁻ and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl⁻ transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell.

     

Measles virus inhibits acquisition of lymphocyte functions but not established effector functions

The effect of measles-virus infection on effector activities of human lymphocytes and on the generation of certain effector activities was studied in vitro. Addition of measles virus to allogeneic mixed lymphocyte cultures resulted in a strongly depressed cytolytic activity in a subsequent cell-mediated lympholysis assay. Late addition of measles virus did not inhibit cytotoxic effector function, although effector cells were probably infected. Similarly, measles-virus infection did not affect the ability of lymphocytes to mediate antibody-dependent cellular cytotoxicity. Addition of measles virus to lymphocytes with, or shortly after, exposure of the cells to the polyclonal activator pokeweed mitogen resulted in abolition of the synthesis of immunoglobulins in vitro. When the virus was added late, the rate of Ig secretion was only partially inhibited. Finally, when lymphocytes were cultured without stimulus in medium supplemented with fetal bovine serum, a population of inhibitory cells was generated. Measles virus was able to prevent the generation of such inhibitory cells. In conclusion, measles virus inhibited acquisition of various effector functions, but the activities of committed lymphocytes were generally not affected.     

Studies on the organization of the α-satellite DNA from African green monkey cells using restriction nucleases and molecular cloning

α-Satellite DNA from African green monkey cells was analysed with restriction nucleases in some detail confirming and complementing our earlier results. With EcoRI and HaeIII (or BsuRI isoschizomer), about 25 and 10%, respectively, of the satellite DNA were cleaved into a series of fragments of the 172 bp repeat length and multiples thereof. To allow studies with fragments of homogeneous sequence unit length, HindIII fragments were covalently joined with the plasmid pBR313. After transformation 19 clones were obtained, containing up to three monomer fragments. Nine of the clones were characterized by digestion with EcoRI. Three of these had cleavage sites for this nuclease in the satellite DNA portion. In the six clones tested with HaeIII no cleavage site was detected in the cloned DNA. The results are discussed in relation to the nucleotide sequence data recently published by Rosenberg et al. (1978) and in the context of random and nonrandom processes in satellite DNA evolution.     

Rotating frame spin-lattice relaxation experiments and the problem of intramolecular motions of peptides in solution

The application of rotating frame spin-lattice relaxtion to the determination of the intramolecular motions in peptides is discussed, and results are presented on the application of ¹³C T_1p to peptide microdynamics in solution. The effective molecular rotational reorientation times at the amide and amino nitrogens may be derived from appropriate data onT_1p of the carbons adjacent to them. We also show by theoretical caculations that ¹H and ¹³C T_1p experiments of suitable ²H and ¹⁵N substituted peptides will allow intramolecular main- and sidechain motions, characterized by times in the range 10^?1–10^?6 sec, to be investigated.     

Cytotoxic activity of lymphocytes. VI. Heterogeneity of cytotoxins in supernatants of mitogen-activated lymphocytes.

Two different lymphotoxins synthesized by human blood lymphocytes stimulated with phytohemagglutin (PHA) are described. The two toxins are called alpha-LT and beta-LT relative to their elution order on gel chromatography. Their m.w. are 75,000 and 45,000 daltons, respectively. Both toxins appear as early as 7 hr after the addition of PHA, with the amount of beta-toxin exceeding that of alpha-LT initially. Both toxins are differentiated from a third toxin (adherent cell toxin, ACT) made by plastic-adherent cells without requiring mitogen-stimulation. Depletion of macrophages or B cells does not affect the synthesis of either lymphotoxin. Monospecific antisera were elicited to alpha-LT. Antisera elicited to beta-LT also neutralized alpha-LT but to a significantly lesser degree. Alpha-LT appears to be the lymphotoxin described by most other workers. Beta-LT is unstable at 37 degrees C which may explain why the low m.w. lymphotoxin has not been described previously.     

Ectogalactosyltransferase studies in fibroblasts and concanavalin A- stimulated lymphocytes

In this communication, we have demonstrated that hydrolysis of the nucleotide sugar can cause errors in the detection of an ectoglycosyltransferase. Spleen cell suspensions can incorporate radioactivity when incubated with labeled UDP-galactose, but all the activity is due to decomposition of the nucleotide sugar and uptake of the free sugar. The fibroblast cell lines can incroporate carbohydrate directly from UDP-galactose. Several criteria are presented with can be used to demonstrate that a nucleotide sugar is the direct carbohydrate donor.