Zinc-deficient Cells Research Articles

Cellular thiols status and the expression of type I collagen in zinc-deficient hepatic stellate cells

Cellular thiols status and the expression of type I collagen in zinc-deficient hepatic stellate cells

Zinc-regulated genes in Saccharomyces cerevisiae revealed by transposon tagging.

The biochemistry of human nutritional zinc deficiency remains poorly defined. To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast. Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described. About half of the genome was examined. Cells exhibiting differences in lacZ expression on low or moderate ( approximately 0. 1 vs. 10 microm) zinc media were isolated and the gene fusions were sequenced. Ribonuclease protection assays demonstrated four- to eightfold increases for the RNAs of the ZAP1, ZRG17 (YNR039c), DPP1, ADH4, MCD4, and YEF3B genes in zinc-deficient cells. All but YEF3B were shown through reporter gene assays to be controlled by a master regulator of zinc homeostasis now known to be encoded by ZAP1. ZAP1 mutants lacked the flocculence and distended vacuoles characteristic of zinc-deficient cells, suggesting that flocculation and vacuolation serve homeostatic functions in zinc-deficient cells. ZRG17 mutants required extra zinc supplementation to repress these phenotypes, suggesting that ZRG17 functions in zinc uptake. These findings illustrate the utility of transposon tagging as an approach for studying regulated gene expression in yeast.

Sudden infant death syndrome: oxidative stress

In studies of oxidative stress in sudden infant death syndrome (SIDS) there were two major findings: (1) During normal post-natal development, there was a gradual decline in the number of Cu/Zn superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) immunoreactive neurons in the hippocampus and parahippocampus gyrus in the brain; (2) The total number of immunoreactive neurons was elevated in SIDS victims compared to age-matched controls in infants 6 months of age and under (1).SOD and neuronal aging and degeneration in the hippocampus and neocortex were features of SIDS, Alzheimer's disease and Down's syndrome. In the SIDS study of infants from 3–6 months of age, the elevation of SOD in SIDS victims was significant, whereas no significant elevation of GSHPx was detected. An imbalance between SOD and GSHPx was said to be crucial in the prevention of toxicity of free radicals (1).Zinc-deficient cells cannot up-regulate gene expression of the scavenger enzymes SOD and GSHPx in cells exposed to high levels of superoxide and hydrogen peroxide (2).GSHPx coupled to reduced nicotine adenine diphosphate (NADPH) regenerating systems via glutathione reductase is virtually able to guarantee an effective protection of biological structures against oxidative attack (22).When the capacity of the cell to regenerate GSH is exceeded - primarily due to an insufficient supply of NADPH – oxidised glutathione (GSSG) is released from the cell and protein synthesis turns off (20).We hypothesize that the increased incidence of aging and neuronal death and increased incidence of SOD and GSHPx reactive neurons in early post-natal development indicates an increased up-regulation of gene expression of scavenger enzymes during high exposure to oxidative stress after birth.GSH-dependent peroxide metabolism is linked to the pentose phosphate shunt via NADPH-dependent glutathione reductase (GR). GSHPx is a selenium containing enzyme which together with catalase (CAT) SOD and vitamin E protects cells in the free radical chain. Zinc upregulates gene expression of these antioxidants.

Zinc deficiency affects cell cycle and deoxythymidine kinase gene expression in HUT-78 cells

Although zinc is known to be involved in cell proliferation and DNA synthesis, the mechanism by which zinc may regulate these processes is not understood. We have studied the role of zinc on cell proliferation and gene expression of a DNA synthesizing enzyme, deoxythymidine kinase (TK), in a T helper human malignant lymphoblastoid cell line (HUT-78). In zinc-deficient and zinc-sufficient media, the cell doubling time (mean ± SD) of HUT-78 was 59 ± 8 hours and 32.6 ± 6 hours, respectively. The effect of zinc was T cell specific, inasmuch as the cell growth of another T malignant lymphoblastoid cell line, MOLT-3 (immature T cells), was not affected by zinc deficiency. Iron, copper, or manganese did not completely correct the cell growth of zinc-deficient HUT-78 cells. TK activity and the relative accumulation of TK-mRNA were significantly decreased in zinc-deficient cells during the G 1 phase of cell cycle in comparison with zinc-sufficient cells. Nuclear run-on experiments and actinomycin-D studies showed that the transcription of TK-mRNA was affected adversely by zinc deficiency. Cell cycle studies showed that more zinc-deficient cells remained in S phase and did not undergo mitosis in comparison with zinc-sufficient cells. In conclusion, our data show that zinc is a T cell-specific growth factor and that a decreased gene expression of DNA-synthesizing enzyme TK in zinc-deficient HUT-78 cells in G 1 phase affected adversely the DNA synthesis in S phase and delayed cell cycle.

The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation.

The yeast Saccharomyces cerevisiae has two separate systems for zinc uptake. One system has high affinity for substrate and is induced in zinc-deficient cells. The second system has lower affinity and is not highly regulated by zinc status. The ZRT1 gene encodes the transporter for the high-affinity system, called Zrt1p. The predicted amino acid sequence of Zrt1p is similar to that of Irt1p, a probable Fe(II) transporter from Arabidopsis thaliana. Like Irt1p, Zrt1p contains eight potential transmembrane domains and a possible metal-binding domain. Consistent with the proposed role of ZRT1 in zinc uptake, overexpressing this gene increased high-affinity uptake activity, whereas disrupting it eliminated that activity and resulted in poor growth of the mutant in zinc-limited media. Furthermore, ZRT1 mRNA levels and uptake activity were closely correlated, as was zinc-limited induction of a ZRT1-lacZ fusion. These results suggest that ZRT1 is regulated at the transcriptional level by the intracellular concentration of zinc. ZRT1 is an additional member of a growing family of metal transport proteins.

Moderate zinc deficiency in rhesus monkeys. An intrinsic defect of neutrophil chemotaxis corrected by zinc repletion.

Experimental animals fed zinc-deficient diets are well known for susceptibility to infections and impaired mitogen response and Ig production. However, the levels of zinc deficiency used have generally been severe, not comparable to human populations, and have not addressed neutrophil function. To address this issue we have studied the effect in rhesus monkeys of a well defined moderately zinc-deficient (MZ) diet on polymorphonuclear leukocyte (PMN) function. Female adult rhesus monkeys were fed either a control (100 micrograms Zn/g) or MZ (2 micrograms Zn/g) diet for 9 mo with quantitation of PMN chemotaxis, and phagocytosis of opsonized yeast. In addition, membrane potential and secretion responses (changes in 90 degrees light scatter) and changes in PMN shape (forward light scatter shifts) were also measured. When compared to the PMN of animals fed control diets, there was a significant reduction in chemotaxis to FMLP of MZ-fed monkey PMN. Although shape change, cell membrane depolarization, as well as phagocytosis were not significantly different among the two groups, the PMN of MZ animals had significantly lower relative loss of orthogonal light scatter (degranulation) due primarily to a lower resting orthogonal light scatter and also a smaller loss when stimulated with FMLP. In vitro addition of zinc to the cells (25 microM) did not improve chemotaxis, and in fact, was inhibitory for most control and zinc-deficient cells. However, after 2 wk of dietary zinc repletion (100 micrograms Zn/g), chemotaxis in the low zinc group was higher and comparable to the control response. These data indicate that zinc deficiency is associated with an intrinsic PMN defect that specifically affects chemotaxis and is corrected with dietary zinc repletion.

Histone formation, gene expression, and zinc deficiency in Euglena gracilis.

Histones, and other basic proteins, have been isolated from zinc-sufficient (+Zn) Euglena gracilis by standard chromatographic methods. These cells contain 2.46 micrograms of histones and 1.96 micrograms of DNA per 10(6) organisms. Each of the histones, H1, H3, H2A, H2B, and H4, is present in both log- and stationary-phase +Zn cells and has been characterized according to its electrophoretic mobility and molecular weight. H1 has been further identified on the basis of its amino acid composition and its cross-reactivity with calf thymus histone H1 antibodies. Similarly, H3 has been recognized as well by its specific reaction with an H3 antibody. In contrast, log-phase zinc-deficient (-Zn) cells contain H1 and H3 while H2A, H2B, and H4 are absent. All of the histones vanish in stationary-phase-Zn organisms. The DNA content increases as the -Zn cells progress from log to stationary phase, reaching a value of 4.40 micrograms/10(6) cells, double that of comparable stationary-phase +Zn organisms. A 2000-3000-dalton polypeptide whose electrophoretic behavior differs from that of the known histones constitutes over 90% of the total basic proteins of -Zn cells. On addition of zinc to stationary -Zn cells, cell division resumes, and all the histones and other basic proteins reappear. Together with previous results, the data demonstrate that zinc significantly affects the metabolism of all major chromatin components, i.e., the RNA polymerases, DNA, and histones of E. gracilis [Vallee, B.L., & Falchuk, K.H. (1981) Philos. Trans. R. Soc. London, Ser. B 294, 185-197]. The implications of these effects of zinc on chromatin structure and function are discussed.

Regulation of human lymphoblast plasma membrane 5'-nucleotidase by zinc.

Human lymphoblasts lose >90% of their plasma membrane 5’-nucleotidase activity within 24 h when grown in zinc-deficient (<0.05 ~LM Zn(I1)) culture media prepared with either bovine serum albumin/fatty acids/ transferrin or with extensively dialyzed fetal bovine serum as supplements. The loss of 5’-nucleotidase activity is accompanied by a decrease in substrate affinity. The inactivation of plasma membrane 5’-nucleotidase is temperature dependent and requires the metabolically active cell. When isolated plasma membranes are incubated under the same conditions as the whole cells, they do not lose their 5‘-nucleotidase activity. Supplementation of the zinc-deficient culture media with 10 p~ zinc prevents the loss of 5’-nucleotidase activity. Addition of 10 p~ zinc to zinc-deficient cells or plasma membranes prepared from cells with low 5’nucleotidase activity due to zinc depletion leads to complete recovery of activity within 24 h. The recovery is not prevented by inhibition of protein synthesis. The reactivation of 5’-nucleotidase shows a dependence on temperature which is clearly distinct from the inactivation process. The addition of Co(II), Cu(II), or Cd(I1) to zinc-deficient cultures leads to some activation of 5’nucleotidase, but none of the divalent cations tested were nearly as effective as zinc. The observed changes in 5’-nucleotidase activity were not found in other divalent cation-requiring plasma membrane enzymes (ATPase, alkaline phosphatase). Our findings suggest that 1) plasma membrane 5’-nucleotidase can exist as an inactive apoenzyme and 2) zinc plays a unique role in the expression of plasma membrane 5’-nucleotidase activity.

Mineral Deficiency Effects on the Generation of Cytotoxic T-Cells and T-Helper Cell Factors in Vitro

Generation of cytotoxic T-cell response of splenocytes was studied in vitro under copper, magnesium and zinc-deficient conditions. Viability of the short-term lymphocyte cultures in the deficient media was comparable with control condition viability. Cell mediated lympholysis (CML) was analyzed in an alloantigen-stimulated mixed lymphocyte culture (Balb/c versus CBA/H mice) using a 51Cr release assay. Lymphocytes cultured in copper and magnesium-deficient media failed to generate specific lysis to allogeneic target cells, whereas lymphocytes cultured in zinc-deficient media did generate T-killer cell activity at reduced levels. In examining the site of mineral deficiency effects, the actions of T-helper cell-produced factors was studied. There was no production of T-cell replacing factor (TRF) in any of the elementally deficient media by cultured splenocytes. The addition of TRF produced under normal control conditions to copper-deficient media completely restored the CML, whereas only a partial restoration of the CML was noted for the magnesium and zinc-deficient cells. The defect in the CML in the copper-deficient media appears to be focused on the T-helper cell, but magnesium and zinc deficiency effects appear to also be at other levels of cell differentiation and proliferation in the generation of CML.Copper magnesium T-cell response zinc

Effect of zinc ions on δ-aminolevulinate dehydratase in Ustilago sphaerogena

The activity of δ-aminolevulinate dehydratase (E.C. 4.2.1.24) in crude extracts of the smut fungus Ustilago sphaerogena, grown in its sporidial stage, has been found to depend on the concentration of Zn ++ in the culture media; half-maximal effect was at about 2 × 10 −6 M. Dialysis of crude extracts did not result in the loss of enzyme activity, which might be expected if the enzyme is activated by reversible enzyme-Zn ++ complex formation, and addition of Zn ++ in vitro had little effect up to 10 −5 m. Higher concentrations of Zn ++ were inhibitory in accord with the implication of essential SH in this enzyme as revealed by inhibition with Cu ++ or iodoacetate. Attempts to obtain highly active enzyme by the addition of Zn ++ to crude extracts from zinc-deficient cells were unsuccessful. The increase in the activity caused by the addition of Zn ++ (3 × 10 −5 m) to a culture previously grown in the presence of low concentration of Zn ++ (10 −7 m) was effectively inhibited by simultaneous addition of p-fluorophenylalanine (4.5 × 10 −4 m), but little inhibition was observed when actinomycin D (200 mg/ liter) was substituted for the amino acid analogue. Thus it appears that Zn ++ is required for the synthesis of this enzyme protein at the translation level.