Over-Expression of an Arabidopsis Zinc Transporter in Hordeum Vulgare Increases Short-Term Zinc Uptake after Zinc Deprivation and Seed Zinc Content

Increasing the zinc content of cereal grains will be important for improving human nutrition. Improved plant zinc efficiency will lead to increased yields when available zinc is limiting plant growth. The aim of our work was to test how the over-expression of zinc transporters in cereals affects plant growth, seed mineral content, and zinc transport rates. Known zinc transporters from Arabidopsis were over-expressed in Hordeum vulgare cv. Golden Promise by means of a ubiquitin promoter. Multiple transgenic lines were obtained, and the locus number and expression levels were verified. Transgenic lines were tested in long-term growth and short-term uptake experiments. Seeds from transgenic lines grown in soil had higher zinc and iron contents than controls. Short-term uptake rates were higher in the transgenic lines after zinc deprivation. Resupply of zinc after a period of deprivation resulted in the rapid decrease in zinc uptake even in transgenic lines in which a zinc transporter gene was constitutively expressed. Similar to processes in yeast and Arabidopsis, we hypothesize that this rapid decrease in zinc transport activity may be caused by the degradation of transporters in response to zinc-sufficient conditions. In the long-term growth experiments, there were no significant differences between transgenic and control lines in leaf zinc content or shoot biomass under zinc-sufficient or -deficient conditions. However, root-to-shoot ratios were higher in the transgenic plants grown under low-zinc conditions; this could impact zinc acquisition under field conditions. Increased seed zinc and iron content by over-expression of a zinc transporter provides a new strategy for increasing the micronutrient content of cereals.     

Zinc and immunity

Nutritional deficiency of zinc is widespread throughout the developing countries and a conditioned deficiency of zinc is known to occur in many diseased states. Zinc is known to play an important role in the immune system and zinc deficient subjects may experience increased susceptibility to a variety of pathogens. We have studied the effects of a mild deficiency of zinc on T cells in an experimental model of human zinc deficiency. We showed that T cell functions were affected adversely even when the deficiency of zinc was mild in humans. Characteristically during zinc deficiency, the serum thymulin activity (a thymic hormone) was decreased which was restored following zinc supplementation. Our studies also showed that zinc deficiency caused an imbalance between TH1 and TH2 functions. The production of IFN-g, IL-2, TNF-a (products of TH1 cells) were decreased, whereas the production of IL-4, IL-6 and IL-10 (products of TH2) were not affected during zinc deficiency. T cell subpopulation studies revealed that the CD4+ CD45RA+ to CD4+ CD45RO+ ratio was decreased as a result of zinc deficiency, suggesting that zinc may be required for the regeneration of new CD4+ T cells. We further documented that zinc deficiency decreased NK cell lytic activity and caused a decrease in the percentage of CD8+ CD73+ T cells which are known to be predominantly precursors of cytotoxic T cells. In a suitable cell culture model our studies revealed that the gene expression of a DNA synthesizing enzyme TK was affected adversely which resulted in delayed cell cycle and decreased cell growth. The above immunological consequences of zinc deficiency may be responsible for decreased cell mediated immune functions in zinc deficient subjects.     

小麦锌营养状况对锌吸收的影响

不论锌的供给形态是无视态(ZnSO_4),还是螯合态(ZnEDTA 或 Zn—PS),小麦锌吸收率均随供锌浓度的提高而增加。缺锌小麦锌吸收率明显高于对照。供ZnEDTA的小麦锌吸收率低于供ZnSO_4和 Zn—PS的小麦。无机态Zn~(24)在根自由空间内累积多,而 ZnEDTA态锌在根自由空间中累积少,缺锌小麦根分泌的有机物质对前者活化量大于后者、被植物吸收利用的数量也就高于后者。     

Psychological Stress-Induced Lower Serum Zinc and Zinc Redistribution in Rats

In humans, long-term exposure to uncontrollable and unpredictable life stressors is a major precipitant in the development of depressive disorders. There are strong evidences that depression is accompanied by lower serum zinc. The aim of present study is to assess the effects of repeated psychological stress (PS) on the zinc metabolism in rat. The rats were divided into control group and PS group which were subdivided into three subgroups: 7-day group, 14-day group, and recovery group (ten rats in each subgroup). PS model was created by a communication box which contains room A and room B. Rats in room A were only exposed to the responses of rats which were randomly given electrical shock for 30 min in room B. PS was given to rats for 30 min every morning for 14 days. The serum corticosterone (CORT), zinc in serum and tissues, and zinc apparent absorption after PS exposure were investigated. The results showed that the serum CORT increased and serum zinc decreased after 7 and 14 days of PS treatment. The zinc concentration in the liver was increased by 14 days PS exposure, whereas its concentration in the hippocampus was decreased by 7 and 14 days of PS exposure. There were no significant changes in zinc concentration in the heart, spleen, kidney, duodenum, cortex, and cerebellum. A decrease in the zinc apparent absorption was observed in the 7- and 14-day PS groups. The increased serum CORT and liver zinc concentrations and decreased serum zinc and apparent absorption of zinc recovered to normal concentrations 7 days away from PS exposure. The results suggest that PS could induce lower serum zinc, which might be correlated with decreased zinc absorption in the small intestine and increased liver zinc accumulation after PS exposure. The consequent effects of decreased hippocampal and serum zinc and increased CORT concentration after PS exposure on stress-related diseases await further research.     

Zinc Z-DNA

Circular dichroism spectra of poly(dG-dC) in the presence of some zinc complexes exhibit the characteristic inversion associated with the formation of a left handed helix. The transition of B to Z DNA is cooperative and slow. The concentration of zinc complex at the mid point of the transition is strongly dependent upon the nature of the ligand bound to zinc. The most efficient species is one with a tetradentate amine for which the mid point is observed at a zinc:nucleotide ratio of 1:24. 31P spectra of one of these complexes confirm the presence of a left handed helix.     

Zinc and autophagy

Autophagy is a highly conserved degradative process through which cells overcome stressful conditions. Inasmuch as faulty autophagy has been associated with aging, neuronal degeneration disorders, diabetes, and fatty liver, autophagy is regarded as a potential therapeutic target. This review summarizes the present state of knowledge concerning the role of zinc in the regulation of autophagy, the role of autophagy in zinc metabolism, and the potential role of autophagy as a mediator of the protective effects of zinc. Data from in vitro studies consistently support the notion that zinc is critical for early and late autophagy. Studies have shown inhibition of early and late autophagy in cells cultured in medium treated with zinc chelators. Conversely, excess zinc added to the medium has shown to potentiate the stimulation of autophagy by tamoxifen, H₂O₂, ethanol and dopamine. The potential role of autophagy in zinc homeostasis has just begun to be investigated. Increasing evidence indicates that autophagy dysregulation causes significant changes in cellular zinc homeostasis. Autophagy may mediate the protective effect of zinc against lipid accumulation, apoptosis and inflammation by promoting degradation of lipid droplets, inflammasomes, p62/SQSTM1 and damaged mitochondria. Studies with humans and animal models are necessary to determine whether autophagy is influenced by zinc intake.     

Zinc Transporter Proteins

Zinc, which is involved in the structure of all enzyme classes, is a micro nutrient element and necessary for growth and development. The ability of zinc to function without causing toxic effects is depends on the protection of its homeostasis. Zinc transporter proteins are responsible for keeping zinc at certain concentrations. Based on their predicted membrane topology, Zn transporters are divided into two major families, SLC39s/ZIPs and SLC30s/ZnTs, which transport Zn in opposite directions through cellular and intracellular membranes. ZIPs increases the zinc concentration in the cytosol. For this, the ZIPs carries the zinc from extracellular and intracellular compartments to the cytosol. ZnTs, reduces the concentration of zinc in the cytosol. For this, ZnTs carries the zinc from the cytosol to extracellular and intracellular compartments. After being transported to the cell, 50% of the zinc is found in the cytoplasm, 30–40% in the nucleus, and 10% in the plasma and organelle membranes. The expression of many zinc transporter proteins in the cell is depending on the concentration of zinc and the physiological problems. The aim of this study is to give information about association of zinc transporter proteins with physiological events and health problems.     

Zinc and Chlamydia trachomatis

Zinc was noted to have significant effects upon the infection of McCoy cells by each of two strains of Chlamydia trachomatis. With a high or low Chlamydia inoculant, the number of infected cells increased up to 200% utilizing supplemental zinc (up to 1 X 10(-4) M) in the inoculation media compared with standard Chlamydia cultivation media (8 X 10(-6) M zinc). Ferric chloride and calcium chloride did not effect any such changes. Higher concentrations of zinc, after 2 hr of incubation with Chlamydia, significantly decreased the number of inclusions. This direct effect of zinc on the Chlamydia remained constant after further repassage of the Chlamydia without supplemental zinc, suggesting a lethal effect of the zinc. Supplemental zinc (up to 10(-4)M) may prove to be a useful addition to inoculation media to increase the yield of culturing for Chlamydia trachomatis. Similarly, topical or oral zinc preparations used by people may alter their susceptivity to Chlamydia trachomatis infections.     

Abundance of Zinc Ions in Synaptic Terminals of mocha Mutant Mice: Zinc Transporter 3 Immunohistochemistry and Zinc Sulphide Autometallography

The mocha mouse is an autosomal recessive pigment mutant on mouse chromosome 10 caused by a deletion in the gene for the delta subunit of the adaptor-like complex AP-3. Based on zinc transporter 3 (ZnT3) immunohistochemistry, zinc TSQ fluorescence and a modified Timm method, previous studies found a lack of histochemically-detectable zinc and a substantial reduction in the ZnT3 immunoreactivity. It has, therefore, been suggested that the mocha mouse could serve as a model for studies of the significance of zinc ions in zinc-enriched (ZEN) neurons. We have chosen the mocha-zinc-model in a study of the significance of ZEN neurons in hypoxia-caused damage in mouse brain. In order to establish that the model was either void of zinc ions or had a significantly decreased level of zinc ions in their ZEN terminals, we repeated the studies that had lead to the above assumption, the only methodology difference being that we used the zinc specific Neo-Timm method instead of the Timm method applied in the original study. We found that, although the ZnS autometallography (AMG) technique revealed a reduction in staining intensity as compared to the littermate controls, there were still plenty of zinc ions in the ZEN terminals, in particular visible in telencephalic structures like neocortex and hippocampus. At ultrastructural levels the zinc ions were found in a pool of vesicles of the ZEN terminals as in the control animals, but additionally zinc ions could be traced in ZEN neuronal somata in the neocortex and hippocampus. The mossy fibres in the hippocampus of mocha mice also bind with TSQ, though less than in the controls. We found ZnS AMG grains in ZEN neuronal somata, which were also immunoreactive for ZnT3. Our study confirmed the decreased ZnT3 immunoreactivity in ZEN terminals of the mocha mouse found in the original study. Based on these findings, we suggest that the mocha mouse may not be an ideal model for studies of the histochemically-detectable zinc ion pool of the central nervous system.     

Zinc and insulin sensitivity

Many studies have shown that zinc deficiency could decrease the response to insulin. In genetically diabetic animals, a low zinc status has been observed, contrary to induced diabetic animals. The zinc status of human patients depends on the type of diabetes and the age. Zinc supplementation seems to have beneficial effects on glucose homeostasis. However, the mechanism of insulin resistance secondary to zinc depletion is yet unclear. More studies are therefore necessary to document better zinc metabolism in diabetes mellitus, and the antioxidant activity of zinc on the insulin receptor and the glucose transporter.     

Interplay Between Autophagy and Zinc

Autophagy is a conserved catabolic process that plays an important role in cellular homeostasis. The study of the interplay between autophagy and zinc has gained interest over the last years. Multiple studies have indicated that zinc stimulates autophagy and is critical for basal and induced autophagy in mammalian cells. Conversely, autophagy is induced by zinc starvation in yeast. There are no studies analyzing the role of zinc in either Microautophagy or Chaperone-Mediated-Autophagy. The mechanisms by which zinc modulates autophagy are still poorly understood. Studies examining loss of function of genes involved in cellular zinc homeostasis have provided novel insights into the role of zinc in autophagy. Autophagy may help cells adapt to changes in zinc availability in medium by controlling zinc mobilization, recycling, and secretion. Zinc is a key player in toxic and protective autophagy.     

Zinc homeostasis and immunosenescence

For more than 50 years, zinc is known to be an essential trace element, having a regulatory role in the immune system. Deficiency in zinc thus compromises proper immune function, like it is observed in the elderly population. Here mild zinc deficiency is a common condition, documented by a decline of serum or plasma zinc levels with age. This leads to a dysregulation mainly in the adaptive immunity that can result in an increased production of pro-inflammatory cytokines, known as a status called inflamm-aging. T cell activation as well as polarization of T helper (Th) cells into their different subpopulations (Th1, Th2, Th17, regulatory T cells (Treg)) is highly influenced by zinc homeostasis. In the elderly a shift of the Th cell balance towards Th2 response is observed, a non-specific pre-activation of T cells is displayed, as well as a decreased response to vaccination is seen. Moreover, an impaired function of innate immune cells indicate a predominance of zinc deficiency in the elderly that may contribute to immunosenescence. This review summarizes current findings about zinc deficiency and supplementation in elderly individuals.     

Zinc in taste function

Zinc has been associated with taste function in humans at several levels of organization—the taste bud, the nerves transmitting taste information, and the brain. Zinc plays specific yet varied roles at each organizational level, although many of these roles have not been clearly identified. They include participation in the structural architecture of the cell, maintenance of cell membrane integrity, and control of activity of several cytoplasmic and membrane enzymes. Early investigators noted that some patients given drugs that altered zinc metabolism or who experienced disease processes associated with abnormalities of zinc metabolism exhibited taste dysfunction. Because of these findings zinc was given to a variety of patients as treatment for taste dysfunction. Initial treatment success was observed, but was quickly tempered by more extensive studies that yielded widely variable results leading to confusion about the role of zinc in both taste function and taste treatment. Further studies revealed that taste disorders were diverse and complex with multiple underlying pathophysiologies that were little understood. Subsequent work by several investigators revealed that patients with zinc deficiency, of any etiology, exhibited taste dysfunction and that treatment of these patients with zinc usually produced improvement of clinical symptoms. These results raised the question of how to define zinc deficiency, for zinc treatment in patients without zinc deficiency was unsuccessful and these patients represent more than three-quarters of all patients with taste dysfunction. New clinical techniques for the definition of human zinc deficiency have been achieved through the use of binding and displacement of⁶⁵Zn on specific sites on erythrocyte membranes; these results offer a guide to the identification of patients (i.e., those with zinc deficiency) who may benefit from zinc treatment.     

Chloroquine Is a Zinc Ionophore

Chloroquine is an established antimalarial agent that has been recently tested in clinical trials for its anticancer activity. The favorable effect of chloroquine appears to be due to its ability to sensitize cancerous cells to chemotherapy, radiation therapy, and induce apoptosis. The present study investigated the interaction of zinc ions with chloroquine in a human ovarian cancer cell line (A2780). Chloroquine enhanced zinc uptake by A2780 cells in a concentration-dependent manner, as assayed using a fluorescent zinc probe. This enhancement was attenuated by TPEN, a high affinity metal-binding compound, indicating the specificity of the zinc uptake. Furthermore, addition of copper or iron ions had no effect on chloroquine-induced zinc uptake. Fluorescent microscopic examination of intracellular zinc distribution demonstrated that free zinc ions are more concentrated in the lysosomes after addition of chloroquine, which is consistent with previous reports showing that chloroquine inhibits lysosome function. The combination of chloroquine with zinc enhanced chloroquine''s cytotoxicity and induced apoptosis in A2780 cells. Thus chloroquine is a zinc ionophore, a property that may contribute to chloroquine''s anticancer activity.     

锌金属蛋白酶家族介绍及结构机理研究进展

锌金属蛋白酶是一类分布广泛,种类繁多的水解酶家族,是近年来人们研究的热点．一般来说,HEXXH保守序列一直都作为锌金属蛋白酶家族分类的依据．除此之外,不同种类的酶还有一些其它的序列特征．谷氨酸锌蛋白（gluzincin）在锌离子配体处共有2组保守区域;甲硫氨酸锌蛋白（metzincin）则拥有1个延长的保守序列HEBXHXBGBXH,里面包含了第3个His配体．大量的金属蛋白酶晶体结构被解析出来,从中可以发现该类酶的活性中心都包含有1个锌离子．锌金属蛋白酶的催化机理通常认为是锌离子与1个水分子结合活化而成．但是,最近的发现证实了并不是所有的锌金属蛋白酶催化都需要水分子参与活化．在本文中,综述了这些已发表的锌金属蛋白酶家族的分类、结构特征、底物特异性识别和催化机理．     

The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity

Background

ZIP5 localizes to the baso-lateral membranes of intestinal enterocytes and pancreatic acinar cells and is internalized and degraded coordinately in these cell-types during periods of dietary zinc deficiency. These cell-types are thought to control zinc excretion from the body. The baso-lateral localization and zinc-regulation of ZIP5 in these cells are unique among the 14 members of the Slc39a family and suggest that ZIP5 plays a role in zinc excretion.

Methods/Principal Findings

We created mice with floxed Zip5 genes and deleted this gene in the entire mouse or specifically in enterocytes or acinar cells and then examined the effects on zinc homeostasis. We found that ZIP5 is not essential for growth and viability but total knockout of ZIP5 led to increased zinc in the liver in mice fed a zinc-adequate (ZnA) diet but impaired accumulation of pancreatic zinc in mice fed a zinc-excess (ZnE) diet. Loss-of-function of enterocyte ZIP5, in contrast, led to increased pancreatic zinc in mice fed a ZnA diet and increased abundance of intestinal Zip4 mRNA. Finally, loss-of-function of acinar cell ZIP5 modestly reduced pancreatic zinc in mice fed a ZnA diet but did not impair zinc uptake as measured by the rapid accumulation of ⁶⁷zinc. Retention of pancreatic ⁶⁷zinc was impaired in these mice but the absence of pancreatic ZIP5 sensitized them to zinc-induced pancreatitis and exacerbated the formation of large cytoplasmic vacuoles containing secretory protein in acinar cells.

Conclusions

These studies demonstrate that ZIP5 participates in the control of zinc excretion in mice. Specifically, they reveal a paramount function of intestinal ZIP5 in zinc excretion but suggest a role for pancreatic ZIP5 in zinc accumulation/retention in acinar cells. ZIP5 functions in acinar cells to protect against zinc-induced acute pancreatitis and attenuate the process of zymophagy. This suggests that it may play a role in autophagy.     

Zinc and its deficiency diseases

The pervasive role of zinc in the metabolic function of the body results from its function as a cofactor of a multitude of enzymes. Zinc is found in every tissue in the body, and because zinc metalloenzymes are found in every known class of enzymes, the metal has a function in every conceivable type of biochemical pathway. Symptoms resulting from zinc deficiency are as diverse as the enzymes with which the metal is associated. If chronic, severe, and untreated, zinc deficiency can be fatal. Less drastic symptoms include infections, hypogonadism, weight loss, emotional disturbance, dermatitis, alopecia, impaired taste acuity, night blindness, poor appetite, delayed wound healing, and elevated blood ammonia levels. Many symptoms of zinc deficiency result from poor diet consumption, but often the most severe symptoms result from other factors including excessive alcohol use, liver diseases, malabsorption syndromes, renal disease, enteral or parenteral alimentation, administration of sulfhydryl-containing drugs, and sickle cell disease. The most severe symptoms of zinc deficiency occur in young children affected with the autosomal-recessive trait, acrodermatitis enteropathica. This disease results in decreased synthesis of picolinic acid which causes an impaired ability to utilize zinc from common food. Because simple laboratory analyses are often not reliable in determining zinc nutriture of a patient, those symptoms caused by suspected zinc deficiency are best verified by the oral administration of zinc dipicolinate. This zinc compound is efficacious and safe and would provide an accurate means of identifying symptoms that do result from zinc deficiency.     

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.     

Zinc uptake and metabolism by hepatocytes

Hepatocytes are in a dynamic equilibrium with the plasma zinc supply. Kinetic analysis of zinc uptake by isolated rat liver parenchymal cells defines two intracellular pools. In one pool zinc is bound relatively weakly and equilibrates rapidly with the medium at 37 degrees C. In the other pool zinc is bound tightly and interacts with the medium slowly at 37 degrees C. Of the two intracellular pools, the slower responding component represents an exchange process with the bulk of total cell zinc. The slow phase of uptake is saturable with albumin in the medium. The smaller pool is in rapid equilibrium with the medium and represents a labile zinc pool that accounts for net zinc accumulation. Both intracellular pools respond to hormonal stimuli. The factors that augment the uptake/exchange of zinc, namely glucocorticoids, glucagon, epinephrine, and dibutyryl cyclic AMP, are also those that stimulate metallothionein gene expression in hepatocytes. Changes in zinc flux into intracellular pools are directly related to the metallothionein content of hepatocytes. Characteristics of the labile zinc pool suggest that it may serve as an initial intermediate in zinc metabolism by hepatocytes as well as more general aspects of liver function related to zinc.