Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.     

Parasitic specialization ofGeotrichum candidum citrus race

Parasitic specialization ofGeotrichum candidum citrus race, the causal agent of citrus sour rot, was investigated. Of seven isolates tested for pathogenecity, all could infect ten species of citrus fruits and edible parts of five species of noncitrus crops. Only one isolate (Ap2), isolated from soil of an apple orchard, could infect apple fruit.     

Effects of photoperiod and aging on locomotor activity rhythms in the onion fly,Delia antiqua

At photoperiods longer than 8h per 24h, adults of the day-active onion fly Delia antiqua showed a major peak of locomotor activity in the late photophase and also bursts of activity induced by lights-on or lights-off. At shorter photoperiods the activity peaks fused. After transfer from long photoperiods to constant darkness (DD), the rhythm free-ran, but only the major peak persisted. This suggests that only the major peak is controlled by the circadian pacemaker. At long photoperiods, the daily phase of the major peak occurred progressively later with age. As a result, the activity at short photoperiods often shifted from photophase to scotophase in old flies. The free-running period (tau) also changed with age; tau was shorter than 24h until 14-20 days after eclosion and thereafter became longer, but a few individuals repeated changes in tau. The phase delay of locomotor activity with age in D. antiqua would be attributable to the increase in tau.     

Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D₂ antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D₂ agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Genetic structure of the IncN plasmid N3

N3, a plasmid of incompatibility group N (IncN) was mapped by cleavage with restriction endonucleases. The restriction fragments were cloned into vector plasmids. All of the genes unique to IncN plasmids such as specific replication machinery, a restriction-modification system, and repair functions were located on a large portion which had no cleavage sites for many of the site-specific six base-identifying restriction endonucleases tested.     

Purification and characterisation of β-N-acetylhexosaminidase from the butter clam, saxidomus purpuratus

A β-N-acetylhexosaminidase [EC 3.2.1.30] has been purified ～98-fold from an extract of the digestive organs of Saxidomus purpuratus by using ammonium sulfate fractionation, and chromatography on Toyopearl HW-50, CM-cellulose, and Sepharose 4B. The purified enzyme, the molecular weight of which was estimated to be ～66,000 by gel filtration, was composed of two sub-units of molecular weight 30,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified enzyme had a pH optimum of 3.8 and an optimum temperature of 55°, and its activity was enhanced ～2-fold in the presence of 0.1m sodium chloride. The Michaelis constants toward p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucoside and -galactoside were 1.2 × 10^?4 and 1.3 × 10^?4m, respectively.     

Chemical ionization-mass spectra of aldobiouronic acids and dialdose dianhydrides

Chemical ionization (c.i.) mass spectra with isobutane as the reagent gas are reported for the peracetates of aldobiouronic acids and related compounds, and for peracetates and permethylated derivatives of dialdose dianhydrides. Ions (M⁺ + 43) having relatively high intensities were detected in the spectra of disaccharides lacking the dianhydride structure. Peracetylated dialdose dianhydrides showed very weak (M⁺ + 43) ions, and permethylated dianhydrides did not show them. The (M⁺ + 43) ion consisted of molecular ion and acetoxyl radical (but not of the reagent gas). In the c.i. mass spectra of the usual disaccharide peracetates, (M⁺ ? 31) and (M⁺ ? 60) ions had large intensities. In contrast, c.i. mass spectra extremely similar to the corresponding e.i. mass spectra were obtained for dialdose dianhydrides.