Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.     

Biosurfactant yields and nutrient consumption of Pseudomonas fluorescens 378 studied in a microcomputer controlled multifermentation system

Production of biosurfactant AP-6 and consumption of carbon (succinic acid) and nitrogen (ammonium ions) by Pseudomonas fluorescens 378 were studied under different growth conditions. The study was performed in a microcomputer controlled multibatch fermentation system which enabled simultaneous running of 10 fermentors. The fermentors were mantled glass vessels, temperature controlled by circulated water, and mixing was arranged by magnetic stirrers. They were connected to the computer system (pH measurement and control) via signal conditioning cards. The microcomputer had a 128 kbytes RAM, two 800-kbyte floppy disc drives, a graphic terminal, and expansion cards. Biosurfactant production was independent of the carbon-to-nitrogen ratio and the phosphorus content in the medium. Omitting the Fe(III) supplement to the medium increased the product yield by 120%. Changes in oxygen transfer rate and pH in the iron deficient cultures did not have any effect on the product yield. Iron deficiency increased the cell consumption of carbon source. Consumption of carbon source in relation to nitrogen uptake (carbon/nitrogen quotient) increased with increasing quotient in the growth medium. The uptake of carbon and nitrogen changed in the intervals of 1.2-1.5 g/g biomass and 0.09-0.16 g/g biomass, respectively. The consumption of carbon increased from 1.5 g/g biomass to 2.0 g/g biomass when the medium concentration of phosphorus was decreased from 0.18 to 0.027 g/L.     

N-acetyl-p-benzoquinone imine-induced changes in the energy metabolism in hepatocytes

The effect of N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, on the energy metabolism in isolated hepatocytes was investigated. Incubation of cells with NAPQI (400 microM) resulted in an immediate uptake into the mitochondria, followed by both reduction and glutathione conjugation of the quinone imine. These reactions were extremely rapid and were associated with depletion of the mitochondrial ATP content (greater than 80% depletion after 1 min exposure). The loss of ATP was accompanied by increases in ADP and AMP, as well as NADP. No effect on mitochondrial NAD was observed during this initial phase. Similar alterations were produced by NAPQI in the cytosolic compartment. Furthermore, incubation of hepatocytes with NAPQI inhibited oxygen consumption by nearly 90% within 10 s. In parallel to these biochemical changes, there was marked bleb formation on the surface of the hepatocytes, which was found to precede cell death (trypan blue uptake). In conclusion, our results demonstrate that during exposure of hepatocytes to NAPQI, dramatic changes in cellular energy metabolism occur. These biochemical alterations may be caused by a rapid decrease in mitochondrial function, and they may play an important role in the initiation of NAPQI-induced cytotoxicity.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

Isolation and characterization of lipid-protein particles containing platelet factor 3 released from human platelets.

Lipid-protein particles with platelet factor 3 measured by the Stypven clotting-time test [Hardisty & Hutton (1966) Br. J. Haematol. 12, 764-776] have been isolated from platelet-release supernatant. Starting material was washed platelets, which were released by treatment with collagen. Purification of the particles from other components in the release material was accomplished by gel filtration on Sepharose CL-4B followed by affinity chromatography on poly-L-lysine-Sepharose CL-4B gel. Chemical characterization showed that the particles were composed of 40% protein, 42% phospholipids, 13% cholesterol and 5% triacylglycerols. The phospholipid composition was 38% phosphatidylcholine, 25% phosphatidylethanolamine, 9% phosphatidylserine, 2% phosphatidic acid and 26% sphingomyelin. No carbohydrate was detected. Electron-microscopic studies revealed the presence of membranous particles with diameters between 70 and 170 nm.     

Inhibition of in vitro granulopoiesis by autologous allogeneic human NK cells

This study demonstrates the ability of human NK cells to inhibit in vitro granulopoiesis of autologous and allogeneic BM cells. NK lytic activity and GM-CFC inhibition was present among nonstimulated lymphocytes from healthy donors and could be increased by treatment of PBL with IFN. Both the cytotoxic NK cells and the GM-CFC inhibitory cells could be enriched for among nonadherent, low-density cells. High-density cells were not cytotoxic, only inhibitory to a small extent, and could become neither cytotoxic nor more inhibitory after IFN treatment. In contrast, low-density cells showed an increased cytotoxic and GM-CFC inhibitory capacity after IFN treatment. The NK mediated GM-CFC inhibition was dependent on cell contact with BM cells, increased with longer preincubation times, and was most efficient against 7-day GM-CFC as compared with 14 day GM-CFC progenitors. In conclusion, these data provide new information about the human NK cell as a potent inhibitor of in vitro granulopoiesis and also as a possible regulator of hematopoiesis in vivo.     

Effect of polyamine on microsomal cytochrome P-450 stimulation of rate and improved coupling of NADPH oxidation to hydroxylation

Elastase-type activities were found associated with lipoprotein fractions in human serum. A metallo-protease hydrolysing Suc(Ala)₃ PNA and soluble elastin peptides was isolated from apoHDL by Sepharose-elastin chromatography. Polyacrylamide gel electrophoresis and immunoelectrophoresis indicated that this elastase-type activity was associated with apoA₁.     

Perchlorate binding to cytochrome c: a magnetic and optical study

1. The effects of perchlorate on cytochrome c have been investigated by 1H and 35Cl NMR, electron paramagnetic resonance and optical spectroscopy. 2. The pK values for the formation and disappearance of the major alkaline conformation were found to be displaced from 9.3 to 8.3 and from 10.4 to 10.9, respectively. The displacement was dependent on the ClO4(-) concentration below 0.1 M. 3. Competition experiments between perchlorate and chloride show that ClO4(-) binds both to the neutral and alkaline forms but with a higher affinity for the latter. The appearance of a new binding site in the alkaline form accounts for the markedly enhanced relaxation rate of 35ClO4(-) in this pH range. Complex formation between cyanide and the alkaline species results in the loss of this binding site, which probably is located close to or within the heme crevice. 4. The neutral form of ferricytochrome c also binds perchlorate strongly as evidence by the unique appearance of a high-spin signal dependent on pH and perchlorate concentration. This signal disappears with the same pK value as the neutral form. The effects of perchlorate on cytochrome c are due to specific binding of this ion.