Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

On the phosphate linkages and the structure of a disaccharide unit of the type-specific polysaccharide of pneumococcus type XIX

The structure of the capsular polysaccharide (S-XIX) of Pneumococcus Type XIX, which contains residues of d-glucose, l-rhamnose, 2-acetamido-2-deoxy- d-mannose, and phosphate, has been investigated by acid hydrolysis, treatment with acid phosphatase, mass spectrometry, and ¹³C-n.m.r. spectroscopy. Phosphoric esters in S-XIX were largely resistant to hydrolysis (4M HCl, 100°, 3 h). With M or 2M HCl at 100° for 3 h, 4-O-(2-amino-2-deoxy-β-d-mannopyranosyl)-d-glucose 4′-phosphate was liberated. More-drastic hydrolysis of S-XIX gave 2-amino-2-deoxy-d-mannose 3-, 4-, and 6-phosphates, and 4-O-(2-amino-2-deoxy-d-mannopyranosyl)-d-glucose and its 4′-phosphate.     

Identification of suberimidate cross-linking sites of four histone sequences in H1-depleted chromatin. Histone arrangement in nucleosome core

The arrangement of 8 histones in the nucleosome core has been investigated by identifying the sites of 4 histone sequences cross-linked with a bifunctional amino-group reagent, dimethyl suberimidate, selected from among 4 diimidoesters of various linker lengths examined. H1-depleted calf thymus chromatin was allowed to react with 14C-labeled suberimidate at pH 8.5 and 0 degrees C. The cross-linked chromatin was then digested exhaustively with trypsin. Almost all the histone fragments were released from the chromatin with 0.25 M HCl and chromatographed on several columns and on paper. Cross-linked peptides were detected by analyzing the content of radioactive suberimidoylbislysine after acid hydrolysis. The chromatographic procedure developed here showed that the whole histone fragments contained 29 mol% of the total linked reagent as suberimidoylbisylsine. The 5 finally purified cross-linked peptides were identified from the total and N-terminal amino acids of each pair of peptides separated by two-dimensional cellulose thin layer chromatography after cutting the linker by ammonolysis. Thus, intramolecular cross-linking was found between Lys-5 and Lys-9 of H2A, and Lys-34 and Lys-85 of H2B, while intermolecular cross-linking was found between Lys-24 (or 27) of H2B and Lys-74 of H2A, Lys-85 of H2B and Lys-91 of H4, and Lys-120 of H2B and Lys-115 of H3 and/or Lys-77 of H4. Most of these lysine residues are located in the DNA-binding segments of the 4 histone sequences identified previously [Kato, Y. & Iwai, K, (1977) J. Biochem. 81, 621--630]. All the 5 or 6 cross-links can be located in a heterotypic tetramer consisting of one molecule each of H2A, H2B, H3, and H4, and a model of the histone arrangement in the tetramer is proposed. Two such tetramers may compose to the histone octamer in the nucleosome core.     

Salmon calcitonin-induced stimulation of 1 alpha,25-dihydroxycholecalciferol synthesis in rats involving a mechanism independent of adenosine 3'':5''-cyclic monophosphate.

The effect of natural salmon calcitonin on accumulation in plasma of 1 alpha,25-dihydroxy-[3H]cholecalciferol from 25-hydroxy[3H]cholecalciferol in vivo was investigated in vitamin D-deficient thyroparathyroidectomized rats into which graded doses of the hormone were continuously infused by use of a balance study system. A dose-dependent increase in plasma concentrations of 1 alpha,25-dihydroxy[3H]cholecalciferol was observed with calcitonin infusion for 6--30h at a rate greater than 20 M.R.C. m-units/h. Infusion of parathyrin or cyclic AMP produced a similar stimulation [Horiuchi, Suda, Takahashi, Shimazawa & Ogata (1977) Endocrinoly 101, 969--974], but the maximal effect of calcitonin was additive to that of either parathyrin or cyclic AMP. Furthermore concurrent infusion of theophylline (0.5 mumol/h) did not potentiate the effect of submaximal doses (3 and 20 M.R.C. m-units/h) of calcitonin. Plasma concentrations of calcium showed a decrease with calcitonin infusion for 30h, but those of Pi remained unchanged. These results strongly suggest that the rat kidney is endowed with a calcitonin-sensitive 1 alpha-hydroxylase system that is separate from the parathyrin/cyclic AMP system and is independent of changes in plasma Pi.     

Volatile emissions of plant tissue cultures

Summary The low-molecular-weight volatiles released by a variety of plant tissue cultures were examined by gas chromatography. Callus cultures invariably produced carbon dioxide, ethylene, acetaldehyde and ethanol. In cultures with developed shoots, ethanol was absent and acetaldehyde was detected only rarely.     

A new assay system of phospholipid exchange activities using concanavalin a in the separation of donor and acceptor liposomes

A new assay system of phospholipid exchange activities is described. The exchange activities were quantitated by measuring the stimulation of phospholipid transfer between two separate populations of liposomes, which contained, as the major constituents, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and cholesterol in molar ratios of 6: 2: 1: 1: 5. One population of the liposomes was made reactive to concanavalin A by the incorporation of 1.8 mol% $\text{α-}\text{d}\text{-mannosyl}\text{-(1 → 3)-α-}\text{d}\text{- mannosyl}\text{-sn-}\text{1,2-diglyceride}$ from Micrococcus lysodeikticus. The concanavalin A-reactive liposomes, a phospholipid donor, were doubly labelled with [6-³H]galactosylglucosyl ceramide and that class of ³²P-labelled phospholipids whose exchange was being measured. The ³H-labelled glycolipid served as a non-exchangeable reference marker. The other population of the liposomes, a phospholipid acceptor, was concanavalin A nonreactive. These two populations of liposomes were incubated with the cytosol protein of rat liver in a total volume of 0.2 ml.After the incubation, two different procedures were used to separate the two liposomal populations. In one procedure concanavalin A was added to agglutinate the reactive liposomes; the flocculated lectin-liposome complex was separated from the non-reactive liposomes by brief centrifugation. In the other procedure the reactive liposomes were trapped by binding to concanavalin A covalently coupled to Sepharose 2B; the complex was separated from the nonreactive liposomes by filtration through a filter paper under suction. In both assay procedures the amount of phospholipid transferred from the donor to the acceptor liposomes was calculated from the decrease of ³²P/³H ratio of the concanavalin A-reactive liposomes during the incubation. By the assay system it is possible to determine phosphatidylcholine and phosphatidylinositol exchange activities in 100 μg of rat liver cytosol protein.     

Methylmercury: Effects on electrical properties of squid axon membranes

At low concentrations (25–100 μM) methylmercury chloride caused a steady increase in the threshold for excitation and on eventual block of action potentials without changing the resting membrane potential in squid giant axons. In the axons exposed to 25 μM methylmercury chloride, peak transient and steady-state conductances were decreased by 58.8 ± 5.1% and 35.9 ± 4.3% (mean ± SEM, 4 axons), respectively and leakage conductance increased to about five times of the control value. Higher concentrations of methylmercury chloride decreased the resting membrane potential. A concentration of 0.5 mM depolarizing the nerve membrane by 16 ± 2 mV (mean ± SEM, 3 axons) in 40 minutes. These changes in ionic conductances and membrane potential were irreversible on washing the axon with drug-free sea water.