Changes in the integrity of large unilamellar vesicles due to their interaction with tobacco cell suspensions

Negatively charged large unilamellar vesicles (LUV) were incubated with tobacco (Nicotiana tabacum var. xanthi) cell suspensions and with the cell-free medium of the cell suspensions. The extent of cell-LUV interaction was determined by the leakage of the LUV contents. Cells enhanced the leakage of LUV contents and this effect increased with cell age. Addition of polylysine to the reaction mixture increased even further the leakage of the LUV contents. The cell-free medium of the cell suspension also affected the integrity of the LUV. Cell-free medium, by itself, promoted leakage of LUV contents and caused a reduction in the leakage exerted by polylysine. Centrifugation (8000g) of the cell-free medium decreased its effect, heat treatment (122°C) did not alter its effect and sonication enhanced it. The effects of the cell-free medium are attributed to the presence of cell wall debris of disintegrated cells.     

Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli

M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P. M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate. Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA. Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions. The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol. Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.     

Teleocidin and phorbol ester tumor promoters exert similar mitogenic effects on human lymphocytes

The tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) affects a wide variety of cellular functions via its binding to protein kinase C (PKC). The TPA molecule contains a diacylglycerol (DAG)-like structure, which may explain its ability to mimic DAG in PKC activation. Teleocidin (TCD) is a different tumor promoter which can compete with TPA in binding to its cell surface receptors even though structurally unrelated to TPA or DAG. Since TCD may use an additional receptor system and/or be distinguished from TPA in its effect on cells, we compared the effects of TPA and TCD on human peripheral blood lymphocytes (PBL). Both tumor promoters preferentially enhanced cell proliferation of sheep erythrocyte-rosetted lymphocytes, which were enriched for T cells. Additionally, TPA and TCD both induced a high density of cell surface receptors for interleukin 2 (IL2) and transferrin, but not synthesis or production of IL2. However, either of the tumor promoters synergized with T cell mitogens to induce high level IL2 production by PBL. In dose response and kinetic studies, matching concentrations of TPA and TCD induced similar effects in PBL. The results thus demonstrate that TPA and TCD are alike in mitogenic capacity, and suggest that structural similarity between the tumor promoter and DAG, the physiological activator of PKC, is not an essential property for promoting tumors or affecting a wide variety of cellular functions.     

Production of interleukin 2 (IL 2) by salivary gland lymphocytes in Sj?gren's syndrome. Detection of reactive cells by using antibody directed to synthetic peptides of IL 2

Because abnormalities in interleukin 2 (IL 2) production have been reported in the blood of patients with certain autoimmune diseases, we have examined the lymphocytes from patients with Sj?gren's syndrome (SS) in which it is possible to obtain simultaneous samples of inflammatory site (i.e., salivary gland) lymphocytes and blood lymphocytes. We found that IL 2 production by peripheral blood lymphocytes (PBL) after mitogen stimulation was markedly diminished (4 +/- 2 U/ml) in 8/32 SS patients. However, salivary gland lymphocytes (SGL) from six out of six SS patients (including three patients with low IL 2 production by their PBL) had a high level of IL 2 production (97 +/- 32 U/ml), suggesting that IL 2 production by inflammatory site lymphocytes may differ from blood lymphocytes in the same patients. Low IL 2 production by a patient's PBL was not correlated with the patient's age, duration of disease, immunoglobulin level, or presence of antinuclear antibodies. Low IL 2 production was associated with a decreased ratio of Leu-3a/Leu-2a positive cells (p less than 0.05) and with an increased proportion of "activated" T cells expressing HLA-DR and gp140 (p less than 0.05). To determine the proportion of PBL and SGL containing cytoplasmic IL 2-like material, we used affinity-purified rabbit antibodies prepared against chemically synthesized peptides of human IL 2. Before mitogen stimulation, PBL were not stained by these antibodies (less than 1% reactive cells), whereas SGL T cells eluted from the salivary gland of SS patients contained a small (3.4% +/- 1.8) proportion of reactive cells. A similar proportion (2.4% +/- 1.2) of reactive cells was noted when frozen tissue sections of salivary gland biopsies were examined with these antibodies. After mitogen stimulation, 35% +/- 17 of PBL and 56% +/- 18 of SS SGL were specifically stained with anti-IL 2 peptide antibodies. In summary, these studies demonstrate a significant difference in IL 2 production between PBL and SGL of the same patients. Furthermore, antibodies against IL 2 peptides provide a powerful tool for detection of T cells producing IL 2 in vitro and in situ, and for understanding the role of this lymphokine in pathogenesis.     

ATP-induced ΔpH formation in chloroplast ATP synthase proteoliposomes

Summary A procedure to reconstitute CF₀CF₁ proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced pH of 2.5 to 3.5 units. pH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF₀CF₁ proteoliposomes prepared by conventional techniques catalyzed an ATP-induced formation, but were unable to catalyze an ATP-induced pH even in the presence of valinomycin.The ATP-induced pH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced pH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF₀CF₁ preparation. The ammonium acted as an internal buffer, and thus prevented an observable pH formation.     

Subcellular localization of lethal lysis proteins of bacteriophages lambda and phiX174.

The gene products of the lethal lysis genes S and E of the bacteriophages lambda and phiX174, respectively, were shown to be associated primarily with inner membrane material by isopycnic sucrose gradient centrifugation of lysates of infected cells. A small amount of each polypeptide appeared to be in the outer membrane fraction.     

Data transfer from Beckman LS 5800 liquid scintillation counters to IBM personal computers

This communication describes a short routine in BASICA for theIBM-PC, written to collect data from a Beckman liquid scintillationcounter. In the form presented here the routine converts incomingbytes into separate lines and saves these lines in a file. Thereare many possible applications for further use of the data inthese files. A few suggestions are given as to the format inwhich data can be stored and how to process these data automaticallyafter all samples have been counted. The only hardware neededis an asynchronous communications adapter for the IBM-PC andan RS232 cable. Received on July 27, 1985; accepted on August 2, 1985     

The Effects of Some Chlorinated Hydrocarbons on Certain Soil Bacteria

S ummary . Some common soil bacteria have been shown to tolerate the presence of chlorinated hydrocarbon fumigants. Several of these bacteria indicated an ability to utilize the chemicals as carbon or energy sources. The breakdown of these fumigants may yield compounds either beneficial or detrimental to plants grown in treated soil. Such information is critical to plant disease control since these chemicals are used for the control of plant pathogens, primarily nematodes. It is suggested that the side effects of soil treatment by which plants are stimulated in their growth is a direct consequence of enhanced beneficial microbial activity.     

μ-Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine-Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons

The receptors mediating the inhibition of D1 dopamine receptor-stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose-dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50 = 0.05 microM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 microM). SKF 38393 (1 microM)-stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective mu-agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO; EC50 = 0.006 microM) and high concentrations of the selective delta-agonist [D-Ser2(O-tert-butyl), Leu5]-enkephalyl-Thr6 (DSTBU-LET; EC50 = 0.13 microM) but not by the selective delta-agonist [D-penicillamine2, D-penicillamine5]enkephalin (DPDPE). D1 dopamine receptor-stimulated adenylate cyclase activity was also slightly reduced (by approximately 20%) by high concentrations of the kappa-agonist U50,488 (EC50 = 0.63 microM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the mu-opioid receptor-selective antagonist naloxone (EC50 of approximately 0.1 microM). Neither the irreversible delta-ligand fentanyl isothiocyanate (1 microM) nor the reversible delta-antagonist ICI 174864 (1 microM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (greater than 0.1 microM) of the kappa-opioid receptor-selective antagonist norbinaltorphimine. The effect of DAGO (1 microM) was already detectable after 1 day in culture, whereas DPDPE (1 microM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of mu-opioid receptors coupled as inhibitors to D1 dopamine receptor-stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.