Induction and degradation of Zn-, Cu- and Cd-thionein in Chang liver cells

Human liver cells (Chang liver) were exposed to 5 micrograms Zn, 2.5 micrograms Cu or 1 microgram Cd/ml in cultured medium. These exogeneous heavy metals were accumulated by the cells and induced de novo synthesis of metallothionein after a 3-h incubation period. The production of Zn-, Cu- or Cd-thionein started in the cells with accumulation of 1 nmol Zn, 0.3 nmol Cu and 0.1 nmol Cd/mg cytosol protein and subsequently the amounts of metal-binding thioneins increased in agreement with the relative amount of metal accumulated in the cytosol over a 24-h period. When cells containing Zn- or Cu-thionein were placed in metal free medium, 70% or 25% of the zinc or copper bound to each original metallothionein was released after 3 h; bound metals decreased to 85% and 65% respectively after 24 h. The disappearance of metal from metallothionein correlated with increases of metal in the medium. On the other hand, 35S-counts incorporated into Zn- and Cu-thionein decreased only to 40% and 15% of the levels in the original metallothionein after 3 h; 35S-counts decreased to 65% and 45%, respectively, after 24 h, indicating that metals bound to metallothionein decreased more quickly than 35S-counts. These results suggest that metals were released from metallothionein and were excreted into the medium. However, 35S- and 109Cd-counts in Cd-thionein changed very little, if at all, in the cells even after a 24-h incubation period. Our data strongly suggest that Zn- and Cu-thionein are degraded in the cells, but that Cd-thionein remains longer than either Zn- or Cu-thionein. When cells containing Zn-thionein were incubated in metal-free medium, Zn-thionein was digested in the cells and peptide fragments ranging about 200-400 daltons were excreted from the cells.     

Overlapping imprinting of oligopeptides in Chang liver cells. Data on the mechanism of hormone evolution

Imprinting was induced with synthetic oligopeptides in Chang liver cell cultures to test these molecules for signal molecule value. Investigations into imprinting overlaps (cross-imprinting) have shown that all oligopeptides (di-, tetra- and pentapeptides) carrying a terminal proline group were able to imprint the cells for the pentapeptide Tyr-D-Met-Gly-Phe-Pro-NH2, which displayed an outstanding imprinting potential for itself and an extraordinary opioid activity as well. The fact that exclusively the proline-deficient oligopeptide (a tetrapeptide) failed to imprint for the pentapeptide in question, indicates a decisive role of proline in the transformation of molecules to signal carriers (hormones). The pentapeptide in question did imprint for the related molecules (except the dipeptide) but to a much lesser degree than for itself. The marked inferiority of the pentapeptide's cross-imprinting potential to its self-imprinting potential supports the hypothetical implication that a considerable difference between the specific and non-specific binding capacities of a molecule, if not the loss of non-specific binding was an essential prerequisite of transformation to a signal molecule, i.e. of hormone evolution.     

Interrelationships between the imprinting potential and biological activity of insulin derivatives: studies on Tetrahymena and Chang liver cells

Insulin dimers deprived of biological activity by linking with suberic acid symmetrically in position B29 or B1 were not able to induce imprinting. Lack of N-terminal phenylalanine or even of five C-terminal amino acids did not interfere with imprinting, regardless of whether or not it was associated with an activity loss. It appears that while hormonal imprinting is closely associated with the hormone's ability to bind to the receptor, it may be related as well as unrelated to the hormone's biological activity. The imprinted Tetrahymena and Chang cells bound the insulin and its derivatives in a similar manner.     

Expression of liver and placental alkaline phosphatases in Chang liver cells

This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase, is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immumochemical assay, ³²P-labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence-activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. Chang liver cells also synthesize a phosphatase which is similar if not indentical to the placental alkaline phosphatase. Since a placental-type alkaline phosphatase has been observed in a number of non-trophoblastic cell lines and also in some neoplasms, it does not seem reliable as an index of the origins of the cell line. Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.     

Biosynthesis of alpha-galactosidase A in cultured Chang liver cells

An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.     

Selective killing of CD8+ cells with a 'memory' phenotype (CD62Llo) by the N-acetyl-D-galactosamine-specific lectin from Viscum album L

As reported previously by our group, among the toxic proteins from Viscum album L. only the mistletoe lectins (MLs) induce the apoptotic killing pathway in human lymphocytes. Although one may expect a homogenous distribution of carbohydrate domains on cell surface receptors for the carbohydrate binding B chains of the toxic protein, the sensitivity of cells to these B chains obviously differ. Here we report a selective killing of CD8+ CD62Llo cells from healthy individuals by the galNAc-specific ML III (and RCA60, which binds to gal and galNAc), while the gal-specific ML I was less effective. This selective killing is not sufficiently explained by protein synthesis inhibition alone, since this subset was not affected by other ribosome inhibiting proteins such as the lectin from Ricinus communis (RCA120), lectin from Abrus precatorus (APA), abrin A, and inhibitors of RNA, DNA and/or protein synthesis such as actinomycin D, mitomycin C, and cycloheximide. We conclude that CD8+ cells with 'memory' phenotype (CD62Llo) are more sensitive to the ML III-mediated killing than their CD8+ CD62Lhi counterparts, CD4+ T cells, and CD19+ B cells. These cells probably express a distinct receptor with galNAc domains that is missing or not active on CD8+ cells with a 'naive' phenotype.     

Induction of cadmium-thionein in isolated rat liver cells.

The uptake of cadmium by isolated liver cells was linearly related to the cadmium concentration to which the cells were exposed in the medium. Cadmium-treated cells synthesized proteins de novo with the characteristics of cadmium-thionein induced in the liver of cadmium-treated animals. Thionein from liver cells incorporated cadmium and [35S]cysteine, had a Ve/Vo (Sephadex G-50) of 1.8-1.9, and was separated into two subfractions by DEAE-cellulose ion-exchange chromatography. Cycloheximide and actinomycin D when added after a cadmium exposure prevented the synthesis of thionein. However, addition of actinomycin D after synthesis had started only decreased the total amount of thionein synthesized. The concentration of cadmium to which the cells were exposed affected the amount of cadmium-thionein synthesized in 6h. The maximum response occurred when cells were exposed to 0.5 microgram of cadmium/ml; at higher metal concentrations the total amount of cadmium-thionein synthesized declined. The system described in the present paper can be used to study the mode of metal toxicity and the mechanism of cadmium-thionein synthesis.     

Ultrastructure of antibody dependent cellular cytotoxicity in HSV 1 infected and uninfected Chang liver cells

The binding and ultrastructural features of antibody dependent cellular cytotoxicity (ADCC) mediated by human peripheral blood lymphocytes were studied in herpes simplex virus type I (HSV-1) infected Chang liver (CL) cells plus human anti-HSV-1 serum, and in uninfected CL cells plus guinea pig anti-CL antiserum. Non-cytolytic controls included target cells treated with normal serum in place of sensitized targets and heat shocked lymphocytes instead of normal lymphocytes. By transmission electron microscopy, target cell membranes were either broadly indented by effector cells or locally invaginated by means of effector cell filopodia. In neither case did the indentation appear to break the plasma membrane of the target. Control preparations showed only non-indented areas of simple membrane contact. By scanning electron microscopy, the effector lymphocytes in both the active ADCC and normal serum control preparations had a sparse distribution of short microvilli over their surfaces. The majority of heat shock control lymphocytes appeared normal, but 12-20% demonstrated surface patches devoid of microvilli. The hypothesis that ADCC may involve a three-step process is discussed.     

Ultrastructural study of biochemically modulated ADCC in HSV-1 infected and uninfected Chang liver cells

The effect of cytochalasin B, ouabain and 25-OH cholesterol on specific lysis due to antibody dependent cellular cytotoxicity (ADCC) in allogeneic and xenogeneic systems was studied using Herpes simplex I infected Chang liver cells. Cytochalasin B reduced both cytotoxicity and lymphocyte/target (LT) binding in the allogeneic system whereas cytotoxicity but not LT binding was reduced in the xenogeneic system. Ouabain inhibited ADCC in both systems as well as LT binding in the allogeneic system; however, binding in the xenogeneic system was not significantly reduced. The 25-OH cholesterol produced a marked decrease in ADCC in both systems but had no significant effect on LT binding in either system. The biochemical and ultrastructural data suggest that the modulators act at different stages in the ADCC response and that there may be more than one mechanism of ADCC to handle different types of target antigens.     

Low intensity microwave radiation effects on the ultrastructure of Chang liver cells

Chang liver cells (CCL-13 ATCC) exposed to 2450 MHz microwaves of field intensities ranging from 5 to 20 mW/cm2 for different periods up to 2 h show distinct alterations in the cytomembrane ultrastructure. A 30-min exposure of 10 mW/cm2 produces well-defined cytoplasmic lesions which appear as clear areas of degenerated rough endoplasmic reticulum (RER). Extensive degeneration of RER along with fragmentation and vacuolation, disorganization of mitochondrial membranes and matrix, increased lysosomal activity, and in some cases disruptions of nuclear membrane are seen in longer exposures. Radiation at 20 mW/cm2 produces significant damage to cell membranes in short exposures and treatments of 30 min and longer exposures lead to total disruption of organized cell ultrastructure. The identity of many organelles is lost as the cells become highly heteropycnotic with numerous cytoplasmic projections. Short exposures of 5 mW/cm2 produce very few noticeable differences in ultrastructure. These results confirm earlier observations that membranes may be the primary targets of microwave radiation in cells.     

Leucine transport coupled to proton movement in membrane vesicles from Chang liver cells

Leucine transport into membrane vesicles obtained from Chang liver cells was stimulated by an inward H+ gradient. The stimulatory effect of the proton gradient on the rate of leucine uptake (1 min) was inhibited by the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. When the vesicles had been preloaded with a high concentration of KCl, addition of valinomycin stimulated leucine uptake by the vesicles, showing that the leucine transport is dependent on potential gradient. Leucine-coupled H+ accumulation inside the vesicles was confirmed by measuring leucine dependent quenching of the fluorescence of 9-aminoacridine added to medium. These results imply that electrochemical gradient of proton can serve as a driving force for leucine transport across the cell membrane and proton movement is coupled to leucine transport.     

Induction of a phosphomannosyl binding lectin activity in Giardia

Giardia lamblia, a protozoan parasite that causes widespread diarrheal disease, expresses a surface membrane associated lectin, taglin, which is specifically activated by limited proteolysis with trypsin, a protease that is present in abundance at the site of infection. When activated, taglin agglutinates enterocytes which are the cells to which the parasite adheres in vivo, and in addition, binds to isolated brush border membranes of these cells. These findings suggest that this lectin may be involved in the host-parasite interaction. Taglin is most specific for terminal phosphomannosyl residues and its binding to red cells is mediated by cell surface phosphate residues. Hemagglutinating activity induced by taglin is most active at pH 6.5 and is dependent on divalent cations. A monoclonal antibody to taglin reacts with the surface membrane of live trophozoites and recognizes a protein of 28/30 kDa in lysates of Giardia trophozoites, by immunoblotting. This finding is confirmed by direct demonstration of lectin activity by erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody.     

Effect of intracellular free Ca2+ on leucine transport in Chang liver cells

Addition of Ca2+ ionophore (A23187) to the medium stimulated the Na+-independent leucine transport in Chang liver cells, increasing the cytoplasmic free Ca2+ concentration, irrespective of the presence or absence of extracellular Ca2+. Anticalmodulin drugs, such as chlorpromazine, trifluoperazine, and W-7, significantly inhibited the leucine transport in the cells. The stimulatory effect of A23187 on leucine transport was completely blocked in the presence of the anticalmodulin drug. Two microtubule disrupting drugs, colchicine and colcemid, significantly stimulated leucine transport. On the other hand, taxol, a microtubule stabilizing agent, decreased the stimulatory effect of colchicine on the leucine transport. These results strongly suggest the involvement of Ca2+ and calmodulin in regulation of Na+-independent leucine transport, possibly through control of assembly and disassembly of the microtubule network.