Zinc-binding proteins (ligands) in brains of severely zinc-deficient rats

Previous studies from this laboratory reported the presence of a metallothionein-like protein in brain with an apparent estimated molecular weight of 13,000–15,000 daltons. The synthesis of this protein, which incorporates large quantity of cysteine, is stimulated following administration of zinc and copper and is blocked by actinomycin D. In this study, we report that the synthesis of this metallothionein-like protein is considerably lower in brains of severely zinc-deficient rats in comparison with pair-fed orad libitum fed groups. Furthermore, incubation of partially purified metallothionein-like protein with⁶⁵Zn and chromatography on DEAE A-25 Sephadex produced similar elution patterns in the three experimental groups. However, the extent of binding of⁶⁵Zn to the metallothionein-like protein from the zinc-deficient rats was significantly (p<0.05) lower than the control groups. On the other hand, the total concentration of zinc in brains of zinc deficient rats did not vary from control groups. Since the synthesis of this metallothionein-like protein is reduced by zinc deficiency and is stimulated following administration of zinc, we postulate that the free pool of zinc may regulate the synthesis of its binding protein in the brain.     

Biological function of metallothionein. VI. Metabolic interaction of cadmium and zinc in rats

The accumulation and depletion of cadmium in liver and kidney metallothionein (MT) and the effects of dietary zinc deficiency on cadmium metabolism were studied in rats. The accumulation of cadmium in liver MT started to plateau after 80 days, but there was a linear accumulation of this element in kidney MT over the entire 300-day experiment. Cadmium in MT fractions was depleted very slowly when rats were changed to a diet without cadmium. The accumulation of cadmium in MT also caused zinc to accumulate in this protein, even in rats fed zinc-deficient diets. However, the reverse situation was found not to be true; zinc did not cause cadmium to accumulate in MT. Dietary zinc deficiency limited the binding of injected¹⁰⁹Cd to MT of liver, but not of kidneys or testes. However, zinc-deficient rats fed cadmium in their diets metabolized cadmium similarly to zinc-supplemented rats, suggesting that zinc deficiency does not limit the ability of cadmium to stimulate MT synthesis.     

Relative zinc-binding activities of ligands in the cytosol of rat small intestine

Relative zinc-binding activities of high-molecular-weight zinc-binding ligand (HMW-ZBL), metallothionein (MT) and low-molecular-weight zinc-binding ligand (LMW-ZBL) in the cytosols of rat small intestines under various experimental conditions were examined. Zinc-binding activities of MT decreased and those of LMW-ZBL increased in the intestinal cytosols from most of the experimental rat groups after incubating at 37 degrees C for 2 hr. The relative zinc-binding activity of MT increased with increasing doses of injected zinc and decreased with orally-administered zinc. Isolated MT did not lose zinc-binding activity during incubation at 37 degrees C for 48 hr, but moved from the MT eluting peak to the LMW-ZBL eluting peak after 1 week.     

Zinc-Binding Sites on Selected Flavonoids

Flavonoids have attracted increased attention due to their broad bioactivities related to health and diseases. Modulating metal homeostasis may play an important role in their bioactivities. Recent studies have suggested that dietary flavonoids may affect zinc homeostasis, uptake, and transport. In this work, the zinc-binding sites on a few selected flavonoids have been investigated by ¹H NMR spectroscopy under physiological relevant pH and the species formed were verified by mass spectrometry. Zinc binding induces distinct changes in the proton resonances on the flavonoid rings, providing useful information to locate the Zn-binding sites. No Zn-binding was observed with flavone which lacks a chelation site. Zinc was found to bind to the 3-hydroxyl-4-keto, catechol, and 5-hydroxyl-4-keto chelation sites of flavonol, 3′,4′-dihydroxylflavone and chrysin, respectively. Kaempferol and myricetin chelate zinc at the 3-hydroxyl-4-keto site while rutin binds zinc preferentially at the 5-hydroxyl-4-keto site. However, morin appears to bind zinc at the 1-ether-2-hydroxyl site.     

Uptake and turnover of65Zn in subcellular fractions of brain of rat under normal and zinc-deficient conditions

After a single injection,⁶⁵Zn is slowly taken up by the brain of the rat to a maximum after 7 d, followed by a turnover phase, with a half-time of about 3 wk. In the brain of rats on a zinc-deficient diet, the⁶⁵Zn content in the brain continued to increase up to 30 d after the injection. The uptake and turnover phases in six different subcellular fractions of the brain showed a pattern similar to that of the whole brain in both the control and zinc-deficient rats. There was no internal redistribution of⁶⁵Zn in the brain under conditions of progressive zinc deficiency. The results are discussed in a model for zinc homeostasis in the brain.     

Zinc uptake by blood cells of rats in zinc deficiency and inflammation

In zinc deficiency, the function of leukocytes is impaired. However, the results of studies on the zinc concentration of blood cells in zinc deficiency are conflicting, probably in part because of technical and analytical problems. The aim of this study was to investigate, under standard conditions, the uptake of⁶⁵Zn-labeled zinc by blood cells, taken from zinc-deficient rats and from rats in which an inflammation is induced. In both conditions, the serum zinc concentration is reduced. In clinical practice, this makes it difficult to determine whether the decrease in serum zinc is the result of a real or an apparent zinc deficiency. In stress, like an inflammatory disease, the decrease of zinc reflects an apparent zinc deficiency because of redistribution of serum zinc into the liver and because of decrease in serum albumin concentration. Over 70% of the serum zinc is bound to albumin. Blood cells from zinc-deficient and control rats were isolated using a discontinuous Percoll gradient and incubated under nearly physiological conditions in a⁶⁵Zn-containing medium. A significant increase in the in vitro uptake of⁶⁵Zn-labeled zinc by the blood cells of zinc-deficient rats was seen: erythrocytes 1.3, mononuclear cells 2.0, and polymorphonuclear cells 2.6 times the control values. During inflammation, no change in⁶⁵Zn-labeled zinc uptake by erythrocytes and mononuclear cells was demonstrated after 2 d, although the serum zinc and albumin concentrations were decreased, but a small but significant increase in zinc uptake by polymorphonuclear cells was observed. This study of⁶⁵Zn uptake in vitro under standard conditions may prove of value for distinguishing in patients real zinc deficiency from apparent zinc deficiency owing to, e.g., stress, although additional experiments should be performed. A part of this study has been presented at the Meeting of The American Gastroenterological Association on May 12–18, 1990, San Antonio, TX, and has been published in abstract inGastroenterology 98 suppl., A423.     

Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts

Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), and detected by autoradiography. A unique 65Zn-binding band was observed from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.     

The effects of zinc deficiency on pancreatic carboxypeptidase activity and protein digestion and absorption in the rat

1. Proteolytic enzyme activities were examined in the pancreas of zinc-deficient and control rats. 2. No change was detected in trypsin-plus-chymotrypsin activity. 3. Carboxypeptidase activity was appreciably lowered in zinc deficiency and returned rapidly to normal on zinc therapy. 4. In experiments in which U-¹⁴C-labelled Chlorella protein was fed no evidence was obtained which suggested that the reduction in carboxypeptidase activity had limited the rate of protein digestion or absorption. 5. The specific activity of pancreatic protein synthesized during these experiments was appreciably lower in zinc-deficient than in control rats. 6. A higher proportion of the total activity present, in each organ examined, was in the non-protein fraction in zinc-deficient rats.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Oral zinc aspartate treats experimental autoimmune encephalomyelitis

The essential trace element zinc plays a critical role in the regulation of immune homeostasis. Zinc deficiency or excess can cause severe impairment of the immune response, which points to the importance of the physiological and dietary control of zinc levels for a functioning immune system. We previously reported that injection of zinc aspartate suppresses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), as well as effector T cell functions in vitro. Among the preferred characteristics of novel therapeutics for the treatment of autoimmune diseases such as MS are oral availability and a tolerable effective dose to minimize side effects. In this study, we investigated whether oral administration of zinc aspartate, an approved drug to treat zinc deficiency in humans, is effective in controlling EAE at clinically approved doses. We show that oral administration of 6 µg/day [0.3 mg/kg body weight (BW)] or 12 µg/day [0.6 mg/kg BW] of zinc aspartate reduces clinical and histopathological signs during the relapsing remitting phase of the disease in SJL mice. The clinical effect in mice was accompanied by suppression of IFN-γ, TNF-α, GM-CSF and IL-5 production in stimulated human T cells and mouse splenocytes in a dose-dependent manner. Furthermore, a large array of proinflammatory cytokines was modulated by zinc aspartate exposure in vitro. These data suggest that administration of oral zinc aspartate may have beneficial effects on autoimmune diseases like MS.     

Tumor-host zinc metabolism the central role of metallothionein

Features of tumor and host zinc metabolism are described. Emphasis is placed on tumor-host interactions. Using the model of the Ehrlich ascites tumor in mice, one clear site of modulation of cellular zinc by the amount of nutrient zinc available in the host is a zinc-binding protein with the properties of metallothionein. The selective depletion of zinc from this protein is correlated with the loss of cell proliferation by tumors injected into zinc-deficient animals. The properties of isolated metallothionein are consistent with a role for it as a reactive pool of intracellular zinc which can be donated to apozinc proteins and other structures. The presence of the Ehrlich tumor in mice also perturbs their distribution of zinc: zinc leaves the plasma and is accumulated by liver in the form of newly synthesized zinc metallothionein. During host zinc deficiency, this redistribution is not observed. This may be caused not only by a lack of mobile plasma zinc, but also by an inhibition of the initiation of this host response at the site of the tumor in the peritoneum.     

Renal metallothionein responds rapidly and site specifically to zinc repletion in growing rats

Metallothionein (MT) is important for heavy metals and free radical protection in the kidney. MT is responsive to zinc and primarily localized within the renal cortex. However, site-specific renal responses to dietary zinc repletion are understudied. The objective of this study was to examine the effects of dietary zinc deficiency and repletion on renal MT concentration and immunolocalization in rats. Weanling male Sprague Dawley rats were randomly assigned to either a zinc-deficient, zinc control, or pair-fed to zinc-deficient group. Half of the zinc-deficient and pair-fed rats were repleted with the control diet ad libitum for an additional 24 h. Renal tissue samples were assessed for total zinc, MT concentrations and MT immunostaining. Dietary zinc deficiency reduced renal zinc and MT concentrations, and attenuated intensity and localization of MT. Dietary zinc repletion for 24 h restored renal zinc and MT concentrations, the latter primarily in the proximal convoluted tubules of the cortex. Concentrations of renal MT, but not zinc, were elevated by diet restriction and MT (μg/mg protein) and partially normalized by 24 h diet repletion. In conclusion, renal MT modification due to zinc deficiency or diet restriction can be rapidly normalized in a site-specific manner with normal dietary zinc intake. The results support a role for MT in kidney homeostasis, in particular at the level of the proximal tubules in the cortex. The speed of MT repletion may have clinical implications for dietary zinc in the treatment of acute and chronic renal pathology due to toxins and free radicals.     

Up-regulation of MDP and tuftsin gene expression in Th1 and Th17 cells as an adjuvant for an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus

The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.     

Characteristics of (Na++K+)-ATPase inBoergesenia forbesii

(Na⁺+K⁺)-ATPase proved to be present in the vegetative thalli ofBoergesenia forbesii (Harvey) Feldmann. The ATPase was extracted with Triton X-100 and partially purified by Sephadex G-150 gel filtration. The enzyme was activated with Mg²⁺ and further stimulated by the addition of K⁺ and Na⁺. It was observed thatp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), iodoacetoamide, copper sulfate, zinc sulfate, lead nitrate and cadmium chloride inhibited the enzyme activity, but ouabain was ineffective, andN,N′-dicyclohexylcarbodiimide (DCCD) did not apparently inhibit the activity, but rather promoted it slightly. The ATPase activity was also shown in the isolated cell wall ofBoergesenia thalli, and the enzyme activity was detected in the wall itself by using electron microscopic methods.     

Overexpression of angiotensin II type 2 receptor promotes apoptosis and impairs insulin secretion in rat insulinoma cells

Proper follicular development is crucial for cumulus-oocyte complex (COC) maturation, ovulation and luteinisation. All these ovarian processes are regulated by finely tuned rapid tissue remodeling that involves hyaluronan and interconnecting hyaladherins-rich extracellular matrix synthesis and its breakdown by various proteinase systems like matrix metalloproteinase (MMP). Disrupted tissue remodeling machinery can result into pathophysiologies like atretic follicular cysts formation in polycystic ovary syndrome (PCOS). In present study, we employ superovulated (SO) and polycystic ovary (PCO) rat models and demonstrate that on contrary to SO, PCO rat ovary illustrates abnormal follicular morphology with differential levels of various ovarian factors [like HA (hyaluronan), TSG-6 (TNF-α-stimulated gene/protein 6), PTX-3 (pentraxin-3), HABP1 (hyaluronan binding protein 1), MMP2 (matrix metalloproteinase), MT1-MMP (membrane type 1-matrix metalloproteinase) and COX2 (Cyclooxygenase-2)] along with hyperactivities of gelatinases (like MMP9 and -2). Besides cultured COC expansion is blocked by anti-HABP1 antibody treatment showing reduced HABP1 expression. Overall, as MT1-MMP has inverse relation with HABP1 level and direct effect on MMP2 activity, the observations from current in vivo and in vitro studies indicate that disrupted ovarian HABP1 along with concurrent altered expression and hyperactivation of related MMPs can lead to abnormal follicular maturation resulting into ovarian dysfunction in PCO rat.     

Zinc-induced metallothionein synthesis by Caco-2 cells

Caco-2 cells possess many morphological and biochemical characteristics of intestinal absorptive cells, including the ability to transport zinc. In the present study, metallothionein (MT) synthesis in response to increased levels of zinc was examined. Increased incorporation of [³⁵S]cysteine into MTs was observed when excess ZnCl₂ was added to the medium. The rate of MT synthesis was found to be concentration dependent. Also, induction of MT synthesis was greater early in the culture, before the cells were fully differentiated. Incubation of the monolayers with ⁶⁵Zn and 200 μm zinc revealed that approximately 50% of the zinc incorporated into the cells was associated with MTs. The remainder was associated with large proteins as well as amino acids and small peptides. Actinomycin D and cycloheximide both inhibited the induction of MT synthesis, suggesting that the newly synthesized MTs are a result of expression of MT genes. Hence, Caco-2 cells, a model of intestinal absorptive cells, may be used to examine the role of MTs in zinc absorption.     

The regulation of glutamate dehydrogenase,nitrite reductase,and nitrate reductase in excised pea roots by nitrite

Both nitrite reductase and nitrate reductase were induced by nitrite, but there were differences in the time course of induction and in the response to different NO₂ ^- concentrations between these enzymes. NH₄ ⁺ depressed the induction of nitrite reductase. NADH₂ dependent glutamate dehydrogenase activity was enhanced by those NO₂-concentrations in the medium at which unmetabolized NO₂ ^- occurred in the roots. NADPH₂ and NAD+ dependent GDh activities were not affected. In vivo modification and (or) in vivo activation were probably responsible for the increase in NADH₂ dependent GDH activity.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

Exogenous EDDS modifies copper-induced various toxic responses in rice

Copper is a micronutrient required for living organisms, but is potentially toxic in excess. EDDS enhances the phytoextraction of many metals, but the underlying mechanism is fully unclear. Exposure of 200 μM Cu²⁺ for 3 days resulted in rice seedling growth inhibition, accompanied by a decrease in plasma membrane H⁺-ATPase activity, and an increase in relative electrolyte leakage ratios, indicating that maintaining of membrane structure integrity is crucial in acclimation of plants to heavy metal stress. In addition, the chlorophyll and carotenoid content was markedly decreased and the level of the mRNA of Cytochrome P450 gene, OsHMA9, the sulfate transporter gene, and the metallothionein-like protein gene was observed to increase in response to Cu stress. Cu treatment also induced a global epigenetic response which is associated with cell nucleus condensation. These physiological, genetic, and epigenetic responses of rice seedlings to excess copper were modified by the addition of EDDS, suggesting that the supply of EDDS in medium containing a high concentration of Cu ions could enhance plant tolerance potential to excess Cu toxicity through alleviating Cu-induced poisonous effects at various levels.