Cyclosporin A regulates the expression of HLA-DR on human monocytes by two different mechanisms.

Cyclosporin A (CsA), but not its nonimmunosuppressive analog cyclosporin H (CsH), inhibited the expression of HLA-DR in human monocytes. Induction of HLA-DR by interferon (IFN)-gamma in fresh monocytes was also inhibited by CsA and not by CsH. However, when monocytes were pretreated with either CsA or CsH for 16 hr prior to the addition of IFN-gamma, HLA-DR expression was increased, probably because of a cyclosporin-induced increase in the number of IFN-gamma receptors. Down-regulation of the HLA-DR mRNA by CsA was found to be dependent on continuous protein synthesis. IFN-alpha also inhibited the IFN-gamma-induced HLA-DR mRNA expression and showed synergy with CsA at low concentrations but not at high concentrations of the drugs. A common mechanistic element in the pathways of CsA and IFN-alpha is proposed.     

Cyst hatching in Anostraca accelerated by retinoic acid,amplified by Calcium Ionophore A23187, and inhibited by Calcium-channel blockers

Cyst hatching, under standardized conditions, of the Anostracan species Thamnocephalus platyurus and Streptocephalus dichotomus was significantly accelerated but not increased by applying the morphogen retinoic acid (RA). Cyst hatching was enhanced but not accelerated by artificially increasing the inflow of Ca²⁺ to the embryonic cells, using Calcium Ionophore A 23 187. Cyst hatching was accelerated and amplified, to a level in excess of the summed effects of each treatment, by a combined application of RA and ionophore. It was inhibited almost quantitatively by the Calcium-channel blockers Nifedipin and Verapamil. The significance of these findings is discussed.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS).

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol min-1 mg-1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridium pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 M. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.     

Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.     

Actin organization and fibrin-clot retractile activity of cultured mouse fibroblasts

Summary It is known that human and animal fibroblasts are able to induce the retraction of a fibrin clot. In the present study the correlation between (i) fibrinclot retractile (FCR) activity, (ii) the number of actin stress-lines in mouse fibroblasts during growth in culture, and (iii) the sensitivity of actin stress-lines to a powerful actin-depolymerizing factor (ADF), present in plasma and serum of humans and laboratory animals was investigated. Fibroblasts at early passages (2–4) were tested for these parameters at various intervals after seeding (24, 96, and 168 h). The number of actin stress-lines was progressively higher, while the sensitivity to ADF action was progressively lower in cells cultured from 24 to 168 h; the FCR capacity was significantly decreased at 168 h. These data suggest that cells containing weakly polymerized and/or stabilized actin are more active than those containing highly polymerized and/or stabilized actin in triggering fibroblast contraction.