Effect of medium copper concentration on the growth,uptake and intracellular balance of copper and zinc in Menkes' and normal control cells

The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through normal to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08–8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.     

Endotoxin-induced zinc accumulation by liver cells is mediated by metallothionein synthesis

Endotoxin induces a decrease in zinc concentration in the serum and an increase in zinc levels in the liver. We have studied whether metallothionein (MT), which is a heavy metal-binding protein, is associated with this phenomenon in vitro. When MT of liver cells is induced by a factor secreted by endotoxin-stimulated macrophages, the cells accumulate zinc from the medium. The temporal accumulation of zinc is correlated with the induction of MT, and the accumulated zinc binds to MT. These results suggest that zinc accumulation by liver cells is mediated by metallothionein produced in response to a macrophage factor, which is elicited by endotoxin.     

Induction of metallothionein synthesis in cultured cells derived from rabbit kidney

Metallothionein (MT) synthesis in rabbit kidney-derived RK-13 cells was studied. In response to Cd2+, RK-13 cells synthesized proteins closely similar in chromatographic and electrophoretic behaviors to the liver MTs induced in Cd2+-injected rabbit. These proteins were specifically immunoprecipitated by anti-mouse liver MT-II serum. The rate of RK-13 thionein (apoprotein of MT) synthesis rapidly increased after exposure to 1 microgram/ml of Cd2+, and reached the maximum in 7 h. The dose-response curve for the synthesis was biphasic; a sharp increase up to 0.5 microgram/ml and a slower increase at higher concentrations. RK-13 cells retained kidney-specific properties in terms of responsiveness of thionein synthesis to inducers; The MTs were inducible also by Zn2+ and probably by Hg2+, but not by dexamethasone. This system would therefore be a useful model in vitro for studying the regulation of MT synthesis in kidney cells.     

Protein synthesis in cells infected with Semliki Forest virus is not controlled by intracellular cation changes

Treatment of BHK cells with 1 microM nigericin results in a 55% decrease in K+ and a 3.3-fold increase in intracellular Na+; protein synthesis under these conditions is depressed by 35%. In BHK cells infected with Semliki Forest virus (SFV), protein synthesis is depressed by 76% 6.5 h after infection; intracellular K+ is unchanged, and intracellular Na+ is increased 1.8-fold at this time. These results suggest that the increase in intracellular Na+ in SFV-infected BHK cells does not adequately account for the decrease in protein synthesis, and makes it likely that an increased Na+ concentration is a consequence, not a cause, of alterations in protein synthesis in virally-infected cells. No evidence was obtained for the purported [Alonso, M. A. and Carrasco, L. (1980) Eur. J. Biochem. 109, 535-540; (1981) Eur. J. Biochem. 118, 289-294; (1981) FEBS Lett. 127, 112-114] ability of 1 microM nigericin to permeabilize' cells.     

Induction of intracellular ATP synthesis by extracellular ferricyanide in human red blood cells

Summary Human red blood cells rapidly convert extracellular ferricyanide into extracellular ferrocyanide. The reaction is enhanced by the addition of a substrate, adenosine. This increase of the rate of reaction is abolished by iodoacetate. The results indicate there is a flow of electrons across the membrane of metabolizing red blood cells. The reduction of extracellular ferricyanide is accompanied by the formation of intracellular ATP. The effect of an uncoupler and of inhibitors of oxidative phosphorylation on this reaction was studied under conditions where the natural rate of ATP synthesis was slightly reduced by 10^–4 moles/liter iodoacetate. ATP formation was found to be inhibited by DNP, cyanide, and, to a lesser extent, by azide. Amytal is ineffective. Ferrocyanide enhances ATP breakdown. The action of DNP requires the presence of the cell membrane. It can probably not be related to a stimulation of the membrane ATPase of Laris and Letchworth, nor can it be explained on the basis of Mitchell's chemiosmotic hypothesis by effects on the passive permeability of the erythrocyte membrane to H⁺ or alkali ions. In contrast to methylene blue and other oxidants, ferricyanide does not stimulate oxygen consumption in adult red blood cells.     

Effect of X-radiation on DNA and histone synthesis in ataxia telangiectasia and normal lymphoblastoid cells

The possibility that the radiosensitivity of lymphoblastoid cell lines from patients with ataxia telangiectasia (A-T) is due to an aberrant content of histones has been examined. The histone pattern of lymphoblastoid cell lines derived from A-T patients was found to be indistinguishable from that obtained from normal individuals. X-ray irradiation led to a greater decrease in cell growth rate in the A-T cells than in the normal cells but was accompanied by a greater decrease of DNA synthesis rate in the normal cells. This difference in radiosensitivity was not reflected in differences in the content or rates of synthesis of histones or of major non-histone proteins in these cells. Reduction in the rate of DNA synthesis was not associated with the appearance of the lysine-rich histone variant H1. We conclude that the hypersensitivity to ionizing radiation in A-T cells is not due to fundamental differences in the composition or synthesis of the major chromosomal proteins.     

Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells

(-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells.     

Induction of IgG and IgE synthesis in normal B cells by autoreactive T cell clones

During the course of generating tetanus toxoid (TT)-specific T cell clones frm an HLA-DR2,7 donor, four clones were obtained which proliferated in the presence of autologous monocytes alone without the addition of TT antigen. This proliferation was specifically inhibited by anti-HLA-DR framework mouse monoclonal antibody, and appeared to be HLA-DR-restricted. Two of the clones proliferated in response to HLA-DR2-bearing monocytes, and the other two clones proliferated in response to HLA-DR7-bearing monocytes. The capacity of these four autoreactive human T cell helper clones to induce IgE synthesis in B cells was studied. All four clones stimulated autologous peripheral blood B cells to synthesize IgE and IgG antibody. Induction of IgE synthesis in B cells by the autoreactive T cell clones followed the same pattern of HLA-DR restriction which governed the proliferative response of these clones. These results suggest that the interaction of autoreactive helper T cells with B cell HLA-DR antigens may be important in the activation of IgE immune responses in humans.     

Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

Background  

The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures.     

Altered copper metabolism in cultured cells from human Menkes' syndrome and mottled mouse mutants

Cultured cells of a variety of different types from human Menkes' syndrome patients and brindled mouse mutants exhibit similarly altered responses to changes in extracellular copper concentration. This suggests that the mutations in the mouse and human are very similar and that mutant gene expression is occurring in many different tissues. Intracellular copper levels are markedly elevated in mutant cells in normal medium and in medium containing a hundredfold higher copper. Some cell lines from heterozygotes possess elevated copper levels. Elevated extracellular copper and zinc are significantly more toxic to mutant cells. Mutant cells exhibit normal rates of uptake of copper-64 over a 10-min period but abnormally high accumulation over 24 hr and low rates of efflux. Menkes' fibroblasts become saturated with copper-64 at lower extracellular concentrations than for normal fibroblasts. These data support the idea of enhanced intracellular binding in mutant cells.This work was supported by grants from the Australian National Health and Medical Research Council, the McPherson/Shutt Trust, and the Apex Foundation.     

Induction of synthesis of Hsp104 of Saccharomyces cerevisiae in heat shock is controlled by mitochondria

Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress. When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism. In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock. Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104, the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria.     

Regulation of metallothionein synthesis in HeLa cells by heavy metals and glucocorticoids

Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins that bind heavy metals. MT induction occurs in liver in response to either heavy metal (Zn++ or Cd++) administration or stress. The synthesis of MT can also be induced by either heavy metals or glucocorticoid hormones in HeLa cells cultured in serum-free medium. Induction of MT by zinc is subject to "desensitization." In contrast, dexamethasone (dex) induction results in a continued elevation in the rate of MT synthesis. The stability of MT is dependent on the availability of metal; consequently, MT induced by dex is degraded much more rapidly (half-life of 11 to 12 hours) than MT induced by elevated zinc levels (half-life of 36 to 38 hours). Removal of either inducer results in biphasic degradation curves, as apothionein and zinc come into balance. In contrast, deinduction kinetics for MT synthesis following removal of the two inducers (zinc and dex) are the same, with a half-life of two and one-half hours. Inhibition of RNA synthesis blocks deinduction after removal of inducer. Induction of MT occurs in a wide variety of species, from blue-green algae to man. This system should provide an excellent model for the comparative biochemistry of regulation of gene expression.     

Comparison of histones in normal and xeroderma pigmentosum lymphoblastoid cells

Histones from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells were compared both quantitatively, qualitatively and for binding affinity for DNA. Electrophoretic examination of the histones showed that all five major histone species were present in both cell groups and that there were no quantitative differences between normal and XPA histones. Binding affinity to [3H] mammalian DNA of the histones was determined. No significant differences were observed in binding of either normal or XPA histones to DNA.     

Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.     

Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection.

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.