Endogenous copper is cytotoxic to a lymphoma in primary culture which requires thiols for growth

Summary With the use of bathocuproine sulfonate, a copper-specific chelator as an indicator, we have demonstrated that copper ions, present as a natural medium constituent are toxic to the growth of a lymphoma in primary culture and are principally responsible for the growth requirement of mercaptoethanol and other thiols. By chelating trace copper normally present in the medium, bathocuproine sulfonate retarded the oxidation of cysteine to poorly utilized cystine, thus permitting its direct utilization by the cells for growth.     

Release of pre-absorbed cadmium from cultured cells by the addition of copper ion

Summary The biological interaction between cadmium and copper has been studied. Cadmium uptake of KB cells was specifically inhibited by the addition of copper ions to the culture medium. Cadmium which had already been taken up by the cells was released following the addition of copper ions.The authors are grateful to Dr. M. Ishizawa for his helpful discussion.     

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.     

Copper utilization during embryogenesis ofPalaemon lamarrei

Summary The pattern of copper utilization during the embryogenesis of the freshwater prawn,Palaemon lamarrei has been described. Throughout the incubation period, lasting for 14 to 16 days, the egg ofP. lamarrei is permeable to salts and against a concentration gradient, it absorbs 11 g of salt from the freshwater medium. Out of this total salt uptake, as much as 0.143 g is due to the absorption of copper. Intake of copper, as a function of incubation time, exhibited a more or less similar trend to that of total salt intake and this can be attributed to the increased synthesis of haemocyanin.     

Measuring cellulose decomposition using Benchkote-paper,for the estimation of soil pollution with copper

Summary Cellulose decomposition in paddy soil polluted by copper was determined by the use of polyethylene-backed absorbent paper (Benchkote) as a source of cellulose. It was shown that the paper is a suitable material for measuring the rate of cellulose decomposition. Correlations were found between copper content in the soil, the growth of rice plants and the cellulose decomposition rate.Acknowledgment. Gratitude is extend to Dr M.H. Martin, Department of Botany, University of Bristol, England, for suggesting the use of Benchkote paper.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Antioxidant and prooxidant roles of copper in Tween 20-induced hemolysis of hamster and pig erythrocytes containing marginal vitamin E

The concentration-dependent effects of copper acting either as an antioxidant or as a prooxidant were examined in vitro using Tween 20-induced hemolysis. When cupric ion concentration was more than 10 M, free copper(II) acted as a prooxidant; both extensive hemolysis and production of unknown thiobarbituric acid-reactive substance occurred in hamster and pig erythrocytes irrespective of vitamin E status. However, when cupric ion concentration was 2–4 M in the incubation medium, copper showed a clear antioxidant activity, reducing both hemolysis and malondialdehyde production induced either by diluted peroxide-containing Tween 20 with ascorbic acid and sodium azide in vitamin E-deficient hamster erythrocytes, or by peroxide-containing Tween 20 in pig erythrocytes containing marginal amounts of vitamin E. Copper(II) is taken up by the erythrocytes, where copper(I)-complexes may contribute to the protection of cells with membrane vitamin E against oxidative radical attack.     

Early effects of copper accumulation on methionine metabolism

Wilson's disease is characterized by longterm hepatic accumulation of copper leading to liver disease with reduction of S-adenosylmethionine synthesis. However, the initial changes in this pathway remain unknown and constitute the objective of the present study. Using the Long Evans Cinnamon rat model, early alterations were detected in the mRNA and protein levels, as well as in the activities of several enzymes of the methionine cycle. Notably, the main change was a redox-mediated 80% decrease in the mRNA levels of the methionine adenosyltransferase regulatory subunit as compared to the control group. Moreover, changes in S-adenosylmethionine, S-adenosylhomocysteine, methionine and glutathione levels were also observed. In addition, in vitro experiments show that copper affects the activity and folding of methionine adenosyltransferase catalytic subunits. Taken together, these observations indicate that early copper accumulation alters methionine metabolism with a pattern distinct from that described previously for other liver diseases.     

Enzymes catalyzing the reduction of aldopentoses and the oxidation of pentitols in Mycobacteria]

M. phlei, grown on synthetic Sauton medium (with 6% glycerol as carbon source), had NADPH- and NADH-aldopentose reductase, as well as NAD-pentitol dehydrogenase activities; some of their properties are studied. These activities are not present in BCG grown on the same medium. All experiments of aldopentose-reductase induction in BCG on a D(+)xylose medium were negative.     

Menkes disease

Menkes disease is caused by mutations in the copper-transporting P_1B-type ATPase ATP7A. ATP7A has a dual function: it serves to incorporate copper into copper-dependent enzymes, and it maintains intracellular copper levels by removing excess copper from the cytosol. To accomplish both functions, the protein traffics between different cellular locations depending on copper levels.The mechanism for sensing the concentration of copper, for trafficking, as well as the details of the mechanism of copper translocation across the membrane are unknown. Received 24 September 2007; received after revision 12 October 2007; accepted 17 October 2007     

Alopecia induced in young mice by exposure to excess dietary zinc

Second generation mice were exposed to normal (50 ppm, Group I) or excess (2000 ppm, Group II) zinc in the maternal diet during gestation and lactation, then weaned and continued on the mother's diet until sacrifice at 8 weeks. Tibia zinc reflected dietary intake. Group II had reduced plasma copper, body weight, and hematocrit; the second coat of hair appeared late and was lighter in color than Group I, possibly as an effect of copper and pigmentation development and hair growth.     

Selective inhibition of reproduction in aminopterin-treated nematodes

Summary Aminopterin was applied to the free-living nematode Caenorhabditis briggsae and subsequent growth was recorded. Nematode populations, containing all developmental stages and selected juvenile stages, were exposed to the drug in both growth-promoting and non-promoting media. It is suggested that aminopterin creates a specific requirement for thymine in thymine-free medium. In otherwise growth-promoting medium, aminopterin-induced thymine deficiency will lead to progressively unbalanced growth and maturation and hence to sterility even after removal of the drug. The omission of essential amino acids from the medium during thymine starvation prevents larval growth and results in better reproduction and faster proliferation in aminopterin-free medium. The 4 juvenile stages exhibit a different response to thymine starvation created by aminopterin.     

Regulation of G1 phase progression by growth factors and the extracellular matrix

Cell cycle progression is regulated by both intracellular and extracellular control mechanisms. Intracellular controls ensure that cell cycle progression is stopped in response to irregularities such as DNA damage or faulty spindle assembly, whereas extracellular factors may determine cell fate such as differentiation, proliferation or programmed cell death (apoptosis). When extracellular factors bind to receptors at the outside of the cell, signal transduction cascades are activated inside the cell that eventually lead to cellular responses. We have shown previously that MAP kinase (MAPK), one of the proteins involved in several signal transduction processes, is phosphorylated early after mitosis and translocates to the nucleus around the restriction point. The activation of MAPK is independent of cell attachment, but does require the presence of growth factors. Moreover, it appears that in Chinese hamster ovary cells, a transformed cell line, growth factors must be present early in the G1 phase for a nuclear translocation of MAPK and subsequent DNA replication to occur. When growth factors are withdrawn from the medium immediately after mitosis, MAPK is not phosphorylated, cell cycle progression is stopped and cells appear to enter a quiescent state, which may lead to apoptosis. Furthermore, in addition to this growth-factor-regulated decision point in early G1 phase, another growth-factor-sensitive period can be distinguished at the end of the G1 phase. This period is suggested to correlate with the classical restriction point (R) and may be related to cell differentiation.     

Alopecia induced in young mice by exposure to excess dietary zinc

Summary Second generation mice were exposed to normal (50 ppm, Group I) or excess (2000 ppm, Group II) zinc in the maternal diet during gestation and lactation, then weaned and continued on the mother's diet until sacrifice at 8 weeks. Tibia zinc reflected dietary intake. Group II had reduced plasma copper, body weight, and hematocrit; the second coat of hair appeared late and was lighter in color than Group I, possibly as an effect of copper and pigmentation development and hair growth.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

Summary We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

Effect of cell proliferation on the condensation of the X chromosome in culture medium of the individual karyotype 49 XXXXX]

The influence of cell proliferation on the condensation of the X chromosome was observed in vitro in human fibroblasts with 49 XXXXX karyotype. The frequency of cells with four Barr-bodies is low during the logarithmic growth phase and increases to 80% when the cells are becoming confluent or, independently of cell contact, when cell growth is arrested in a medium with low serum content. The condensation of th X chromosomes is reversible when the cells start growing again in medium with a higher serum content.     

Calcifying bacteria and carbonic anhydrase]

Carbonic anhydrase activity was measured on bacteria known for their calcifying power. The results obtained allow us to conclude that carbonic anhydrase is not present in their enzymatic equipment. Nevertheless, the addition of carbonic anhydrase in culture media increases the growth of the cultures and their calcifying power. This result is due to the direct action of the enzyme on the pH of the culture medium. The results obtained could be applied to the mechanism of dental plaque calcification.     

New concepts in regulation and function of the insulin-like growth factors: implications for understanding normal growth and neoplasia

The insulin-like growth factors (IGFs) are a ubiquitous family of growth factors, binding proteins and receptors that are involved in normal growth and development. They are also implicated in numerous pathological states, including malignancy. IGF-II is a commonly expressed growth factor in many tumors and may enhance tumor growth, acting via the overexpressed IGF-I receptor, a cell-surface tyrosine kinase receptor. The IGF-I receptor may be overexpressed due to mutations in tumor suppression gene products such as p53 and WT-1 or growth factors such as bFGF and PDGF. Thus, this family of growth factors, especially the IGF-I receptor, may present an excellent target for new therapeutic agents in the treatment of cancer and other disorders of excessive cellular proliferation.