Quantitative contribution of the acid production to the intracellular acidification in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O₂ ^–) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O₂ ^–, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/10⁶ cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO₂ in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/10⁶ cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.Abbreviations fMLP N-formyl-methionyl-leucyl-phenylalanine - BCECF-AM 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester - PMA phorbol 12-myristate 13-acetate - CGD chronic granulomatous disease - HMP hexose monophosphate - pHi intracellular pH     

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

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     

Muscle determinants in the ascidian egg are inactivated by UV irradiation and the inactivation is partially rescued by injection of maternal mRNAs

The ascidian egg contains cytoplasmic determinants that specify the fate of larval muscle cells. In a previous study, we developed an experimental system to identify the molecular nature of muscle determinants, in which unfertilized Ciona savignyi eggs were fragmented into four pieces by centrifugation. When inseminated, only nucleated fragments (red fragments) develop into partial embryos that only show differentiation of epidermal cells. One type of enucleated fragment (black fragment) has the remarkable ability to promote muscle differentiation when fused with red fragments. In the present study, using this experimental system, we investigated the molecular nature of muscle determinants. UV irradiation of black fragments suppressed the ability to promote expression of the muscle-specific protein, myosin heavy chain. The wavelength of UV light responsible for the inactivation (250–275 nm) suggested that UV-sensitive targets are nucleic acids. Injection of poly(A)⁺ RNA isolated from an un-irradiated black-fragment-rich fraction into UV-irradiated black fragments partially recovered the ability to promote the expression of myosin heavy chain protein. Poly(A)⁺ RNA from a red-fragment-rich fraction did not rescue the suppression of UV-irradiated black fragments. These results suggest that maternal mRNAs enriched in black fragments are closely associated with muscle determinants in the ascidian egg.     

Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX ( r epeats in t o x in) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with non-overlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.     

Periodic appearance and disappearance of microvilli associated with cleavage cycles in the egg of the ascidian, Halocynthia roretzi

The surface of eggs of the ascidian Halocynthia roretzi, observed with SEM, is essentially smooth until immediately before cell division when numerous microvilli appear and remain during cytokinesis. As the dividing blastomeres become closely adherent, however, the microvilli disappear and the eggs recover their smooth surface. This periodic appearance-disappearance of microvilli is repeated at each cleavage cycle up to at least the 32-cell stage. During blastomere adhesion, microvilli that have appeared near the plane of the first cleavage or of the bilateral symmetry seem to fuse together across the plane to form a zipper-like complex of cytoplasmic processes, which might be responsible for attachment of the two halves of these bilaterally symmetrical embryos via the blastomeres bordering the plane of symmetry.     

Changes in Ethylene Production and in 1-Aminocyclopropane-1-carboxylic Acid (ACC) and Malonyl-ACC Contents of Cocklebur Seeds during Their Development

Ethylene production in developing cocklebur (Xanthium pennsyluanicumWallr.) seeds peaked when the dry weight of the seeds beganto increase in the early period of development. The productionthen began to decrease and stopped when the dry weight increasewas completed. The upsurge of ethylene production in the earlydevelopmental period paralleled increases in ACC synthase activityand the 1-aminocyclopropane-1-carboxylic acid (ACC) contentof the seeds, both of which rapidly decreased later. Malonyl-ACC (MACC) accumulated in developing cocklebur seedsduring the early period of development, before the ACC contentand ethylene production increased. Although the ACC synthaseactivity, ACC content and ethylene production showed markeddecreases, the MACC content remained almost unchanged duringthe middle period of seed development, with a pronounced decreaseoccurring in the late period. Exogenous application of MACCdid not promote ethylene production of seeds collected at thelate developmental stage. Aminoethoxyvinylglycine, an inhibitorof ACC synthase, strongly inhibited the ethylene productionof the same lot of seeds. Therefore, the decrease in the MACCcontent in developing cocklebur seeds was not due to reuse ofMACC for ethylene production. (Received May 24, 1984; Accepted August 15, 1984)     

Levamisole in postoperative adjuvant immunochemotherapy for gastric cancer

Summary The usefulness of LMS in postoperative immunochemotherapy of gastric cancer was investigated. In compliance with the protocol, MMC was given at a dose of 20 mg on the day of gastrectomy, and an additional 10 mg on the next day IV. The patients receiving 600 mg Tegafur daily were then divided into two groups according to whether LMS was also given or not. LMS was administered for 3 days before the operation in a daily dose of 150 mg and for 1 year or more after operation according to a schedule of 3 days' administration followed by an 11-day interval. The 2-year follow-up demonstrated that in stage III patients, the LMS (+) regimen was superior to the LMS (–) regimen, since the former prolonged the relapse-free interval significantly. The survival rate for stage III disease was also significantly higher in the LMS (+) than in the LMS (–) group. There was no significant difference in the incidence of subjective or objective side-effects between two groups. The incidence of agranulocytosis was comparable in the two groups.Gastrointestinal Cancer Research Group, Japan Levamisole Research AssociationChairmen of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationController of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationMembers of the Data Collection and Analysis SubcommitteeThis study was carried out by the Gastrointestinal Cancer Research Group, Japan LMS Research Association (directed by Prof. Kiyoshi Inokuchi, Dept. of Surgery, Kyushu University and Prof. Eiro Tsubura, Dept. of Internal Medicine, Tokushima University). The results were presented in part at the 19th General Meeting of the Japanese Society for Gastroenterological Surgery in February, 1982