Pharmacological doses of Zn<Superscript>2+</Superscript> induce a muscarinic cholinergic supersensitivity

The goal of this study was to evaluate the effect of chronic Zn²⁺ administration (1 mg/kg/day for 1 month) in Sprague-Dawley rats (n=11) on motility and rearing behaviors (number of events/10 min measured in motility cage), on memory (percentage of failures using a footshock double T maze), on the number of muscarinic receptors (using [³H]-QNB as a marker) and on the cholinacetyltransferase (Chat) activity (determined by Fonnun's method) in various brain areas (striatum, hippocampus and frontal cortex), as compared with saline-treated rats (n=10). Our results showed that Zn²⁺ induced a decrease in rearing (control: 24.6±3; Zn²⁺: 15.91±2.19) and in locomotor activity (control: 37±3.79; Zn²⁺: 25±4.37), a decrease in failures during memory trials (control: 26.12±5.6; Zn²⁺: 5.33±2.71) and an increase in muscarinic receptor density (fmol/mg) in the striatum (control: 539±6.18; Zn²⁺: 720±14.69), hippocampus (control: 396±7.41; Zn²⁺: 458±5.05) and frontal cortex (control: 506±10.28; Zn²⁺: 716±16.54). Chat activity (pmol/mg/min) was decreased only in the striatum (control: 4,240±158; Zn²⁺: 2,311±69). We conclude that Zn²⁺ induces a cholinergic functional supersensitivity which is related to receptor upregulation.     

Ribonuclease production of Rhizopus oryzae IFO 4697 under metal ion stress in liquid culture

 Rhizopus oryzae IFO 4697 was found to produce intracellular ribonuclease (RNase), and its growth and activity could be regulated under selected metal ion stress. The addition of Fe²⁺, Mg²⁺ and Zn²⁺ to the SLSR medium was essential to growth and RNase production. Ca²⁺ and Mo⁶⁺ stimulated RNase production. It is concluded that the addition of 100 mg/ml Ca²⁺, 5 mg/ml Mo⁶⁺, 0.7 mg/ml Zn²⁺, 2 mg/ml Fe²⁺, and 49 mg/ml Mg²⁺ to the SLSR medium was the best condition for producing RNase in high specific activity (3780 U/mg protein). This result indicates that a metal ion-regulated liquid medium is an efficient culture method for RNase production. Received: July 19, 2001 / Accepted: April 8, 2002     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

Enhanced Susceptibility to Spontaneous Seizures of Noda Epileptic Rats by Loss of Synaptic Zn2+

Zinc homeostasis in the brain is associated with the etiology and manifestation of epileptic seizures. Adult Noda epileptic rats (NER, >12-week-old) exhibit spontaneously generalized tonic-clonic convulsion about once a day. To pursue the involvement of synaptic Zn²⁺ signal in susceptibility to spontaneous seizures, in the present study, the effect of zinc chelators on epileptogenesis was examined using adult NER. Clioquinol (CQ) and TPEN are lipophilic zinc chelotors, transported into the brain and reduce the levels of synaptic Zn²⁺. The incidence of tonic-clonic convulsion was markedly increased after i.p. injection of CQ (30–100 mg/kg) and TPEN (1 mg/kg). The basal levels of extracellular Zn²⁺ measured by ZnAF-2 were decreased before tonic-clonic convulsion was induced with zinc chelators. The hippocampal electroencephalograms during CQ (30 mg/kg)-induced convulsions were similar to those during sound-induced convulsions in NER reported previously. Exocytosis of hippocampal mossy fibers, which was measured with FM4-64, was significantly increased in hippocampal slices from CQ-injected NER that did not show tonic-clonic convulsion yet. These results indicate that the abnormal excitability of mossy fibers is induced prior to epileptic seizures by injection of zinc chelators into NER. The incidence of tonic-clonic convulsion induced with CQ (30 mg/kg) was significantly reduced by co-injection with aminooxyacetic acid (5–10 mg/kg), an anticonvulsant drug enhancing GABAergic activity, which did not affect locomotor activity. The present paper demonstrates that the abnormal excitability in the brain, especially in mossy fibers, which is potentially associated with the insufficient GABAergic neuron activity, may be a factor to reduce the threshold for epileptogenesis in NER.     

High temperature promotes the inhibition of Zn2+ to physiological performance of green tide-forming seaweed Ulva prolifera

Since 2007, Ulva prolifera-induced green tide occurred every year in the offshore waters of the Yellow Sea in China, which have resulted in large economic loss and heavy damage to local marine ecosystems. In addition, ocean warming and heavy metal pollution have become two main marine environmental issues in the world. However, the interactive effects of ocean warming and zinc (Zn²⁺) exposure on macroalgae remain poorly studied. An experiment was conducted to determine the relative growth rate (RGR) and photosynthetic performance at different temperatures (15, 20, 25 °C) and Zn²⁺ concentrations (0, 0.0026, 0.026, 0.26, and 0.52 mg/L). Results showed that low temperature (15 °C) increased the RGR under the medium levels of Zn²⁺ (0.026 mg/L) compared with high temperature (20 and 25 °C). On the other hand, at 20 and 25 °C the inhibition of Zn²⁺ on the PSII quantum yield and electron transport rate of U. prolifera was promoted. Furthermore, dark respiration rate increased with increases in temperature and Zn²⁺ concentration, while at the high temperature, the ratio of the net photosynthetic rate and dark respiration rate were (P_n/R_d) inhibited, and the inhibition was positively related to the Zn²⁺ concentration at ≥0.26 mg/L. in addition, the photoprotective ability was hindered under high temperature (20 and 25 °C) and the potential photosynthetic ability was restricted under higher levels of Zn²⁺ concentration. We conclude that ocean warming could promote the inhibition effects of heavy metal pollutions on physiological performance of U. prolifera, and probably other marine microalgae as well, on which future studies shall be conducted     

Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding site

It has been reported that Zn₇-metallothionein (MT), contains one weak binding site for Zn²⁺. To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn²⁺. Contrary to the previous study, no evidence was found for Zn²⁺ stoichiometrically bound to the protein with an apparent stability constant of about 10⁸. Indeed, stability constant measurements based upon competition between Zn₇-MT and ligands of known stability with Zn²⁺ showed that all of the protein bound Zn²⁺ displayed the same stability constant at pH 7.4 and 25 °C of (1.7 ± 0.6) × 10¹¹. Brief reaction of Zn₇-MT with strong acid converted it into MT^* and upon reneutralization into Zn₇-MT^*, which demonstrated reactivity of about 1 Zn²⁺/mol MT with competing ligands. Acid titration of Zn₇-MT to pH 2 or below rapidly resulted in the formation of Zn₇-MT^* that displayed biphasic titration with base, revealing the rebinding of lower affinity Zn²⁺ between pH 5 and 7. Since MT is commonly acidified during preparation, care must be taken to document which form of the protein is present in subsequent experiments at pH 7.     

Transient fluctuations of intracellular zinc ions in cell proliferation

Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn²⁺). By employing a fluorescent Zn²⁺ probe, FluoZin-3 acetoxymethyl ester, intracellular Zn²⁺ concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn²⁺ concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn²⁺ concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn²⁺ concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn²⁺ concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn²⁺ concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn²⁺ concentrations, suggest a role for Zn²⁺ in the control of the cell cycle. Interventions targeted at these picomolar Zn²⁺ fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.     

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: High-affinity Zn-binding site at the P-side of the membrane

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn²⁺. Inhibition of liposome-bound bovine cytochrome oxidase by external Zn²⁺ titrates with a K_i of 1 ± 0.3 μM. Presumably, the Zn²⁺-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu_B is tentatively proposed to be responsible for the effect.     

The protective effect of thromboxane synthetase inhibitor UK 38485 against bile duct ligation induced liver injury

In order to elucidate the relation between tissue eicosanoids and liver injury due to bile duct obstruction, we have examined the effects of iloprost, a stable analogue of prostaglandin I₂ (PGI₂), and UK 38485 (UK), an inhibitor of thromboxane synthetase, on prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) in guinea pig liver. 56 male guinea pigs were divided into the following groups: (i) sham operations (SHAM), (ii) bile duct ligated (BDL) group, (iii) guinea pigs given UK (5 μg/kg body wt intraperitoneally 10 min, 8 h and 16 h after bile duct ligation), and (iv) guinea pigs treated with iloprost (ILO) (2 μg/kg body wt intraperitoneally 10 min, 8 h and 16 h after bile duct ligation). Liver damage was assessed by blind quantitation of liver cell necrosis. Bile duct ligation caused an increase in tissue PGE₂-like activity and a decrease LTC₄-like activity. But the most pronounced elevation of PGE₂ was observed in ILO treated group. The LTC₄-like activity level improved significantly in the UK-treated BDL group compared with the BDL only and ILO treated animals. Also, UK was found to be beneficial in preventing the liver cell necrosis due to cholestasis. It is concluded that the ratio of PGE₂/LTC₄ in liver is a valuable marker for cholestatic injury.     

Effect of Nutrition Factors on the Synthesis of Superoxide Dismutase, Catalase, and Membrane Lipid Peroxide Levels in Cordyceps militaris Mycelium

Effect of carbon, nitrogen, and metal ion sources on superoxide dismutase (SOD), catalase (CAT) activities, and lipid perioxide (LPO) levels in Cordyceps militaris mycelium were investigated at stationary growth phase by step supplementing with these nutrition factors in shake-flask cultures. Mycelium was cultivated in several growth media containing different carbon sources. The observed highest SOD and CAT activities were 44.3 U/mg protein in the presence of 20% potato broth plus 2% glucose medium and 93.7 U/mg protein in presence of 20% potato broth plus 1% glucose medium, respectively. By supplementing with either yeast extract or tryptone in 0.1–0.5% concentration range, the highest SOD and CAT activities were 21.1 U/mg protein in medium supplemented with 0.1% yeast extract and 20.7 U/mg protein in medium supplemented with 0.1% tryptone, respectively. Supplementing with Cu²⁺, Zn²⁺, and Mn²⁺ caused a stimulation of SOD synthesis. The minimum LPO level was observed at media presented Zn²⁺. The time course of SOD and CAT biosynthesis showed two maxima, which correspond to the maximum of biomass. High SOD levels and low LPO levels in the medium described above indicated that the appropriate metal ions could provide a suitable protection for cells against oxygen radical damage.     

GPR39 up-regulation after selective antidepressants

Recent studies indicated that zinc activates neural transmission via the GPR39 Zn²⁺-sensing receptor. Preclinical and clinical studies demonstrated the antidepressant properties of zinc. To investigate whether the GPR39 receptor is involved in the mechanism of antidepressant action, we measured the expression of the GPR39 receptor (Western Blot) in the frontal cortex of mice treated intraperitoneally with imipramine (30 mg/kg), escitalopram (4 mg/kg), reboxetine (10 mg/kg) or bupropion (15 mg/kg) for 14 days. The present study shows the up-regulation of the GPR39 receptor protein level after escitalopram (by 290%), reboxetine (by 816%) and bupropion (by 272%), but not imipramine treatment. This is the first report to indicate the involvement of the GPR39 Zn²⁺-sensing receptor in the antidepressant effect of selective monoamine reuptake inhibitors.     

Effects of heavy metals Cd2+, Pb2+ and Zn2+ on DNA damage of loach Misgurnus anguillicaudatus

The effects of heavy metals Cd²⁺, Pb²⁺ and Zn²⁺ at 0.05, 0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1–35 days exposure were examined by single cell gel electrophoresis (SCGE). For each test group, 20 loaches with similar body size (5.17–7.99 g; 11.79–13.21 cm) were selected and kept in aquaria with dechlorinated water at (22±1)°C and fed a commercial diet every 48 h. According to the percentage of damaged DNA with tail and its TL/D (tail length to diameter of nucleus) value, the relationship between DNA damage degree and heavy metal dose and exposure time was determined. Results showed that the percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time. The highest percentage (84.85%) of damaged DNA was shown in 5.0 mg/L Zn²⁺ group after 28 days exposure and the biggest TL/D value (2.50) in all treated groups after 35 days exposure. During the first treated week, the damnification of DNA was mainly recognized as the first level, after that time, the third damaged level was mostly observed and the percentage of damaged DNA was beyond 80%. The joint toxic effects among Cd²⁺, Pb²⁺ or Zn²⁺ revealed much complexity, but it generally displayed that the presence of Cd²⁺ could enhance the genotoxicity of Pb²⁺ or Zn²⁺. In conclusion, the results suggested that there was a significant time-and dose-depended relationship between the heavy metal and DNA damage in hepatopancreas of loach, and SCGE could represent a useful means to evaluate the genotoxicity of environmental contamination on aquatic organisms. __________ Translated from Acta Hydrobiologica Sinica, 2006, 30(4): 399–403 [译自: 水生生物学报]     

ICPBCZin: a red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

A new fluorescent Zn²⁺ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn²⁺ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3′,2′:3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn²⁺ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn²⁺ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn²⁺ probe.     

Alteration of metal ions improves the activity and thermostability of aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Recombinant l-aminoacylase (PhoACY) from a hyperthermophilic archeon, Pyrococcus horikoshii, is a zinc-containing metalloenzyme. When the zinc was substituted by Mn²⁺ or Ni²⁺, its specific activity was significantly increased with acetyl-l-methionine as a substrate. The thermostability of PhoACY was improved when it was incubated with 1 mM Zn²⁺, Mn²⁺ or Ni²⁺. The enzyme with external Zn²⁺ addition had no significant loss of the activity when held at 90°C for up to 12 h and moreover had more than a 10-fold longer half-life even at 100°C, compared to the enzyme without Zn²⁺ addition. A thermostable structure of the enzyme associated with zinc binding is described based on differential scanning calorimetry.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca²⁺]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca²⁺]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.     

Zinc induces DNA damage in tobacco roots

We applied the alkaline version of the single-cell gel electrophoresis (comet assay) to seedlings of heterozygous tobacco (Nicotiana tabacum L. var. xanthi) treated with zinc acetate dihydrate (20 to 80 mM Zn²⁺ for 2 h or 2 to 12 mM Zn²⁺ for 24 h). A dose dependent increase in DNA damage expressed by the tail moment values were observed in nuclei isolated from the roots after 2 and 24 h Zn²⁺ treatments. In contrast, Zn²⁺ did not induce significant DNA damage to leaf nuclei, with the exception of 10 or 12 mM Zn²⁺ for 24 h. Somatic mutations, identified as dark green, yellow, and dark green/yellow double sectors on the pale green tobacco leaves were not detected after any Zn²⁺ treatments. The accumulation of Zn in roots and shoots was determined by inductively coupled plasma optical emission spectrometry and the Zn content in roots was about three times higher than in shoots.     

Effect of flow rate on heavy metal accumulation by rotating biological contactor (RBC) biofilms

Immobilized biofilms are effective in heavy metal removal. The current studies investigated the use of rotating biological contactor (RBC) biofilms in treatment of a wastewater containing cadmium, copper and zinc, each at a concentration of 100 mg L⁻¹. In particular, the influence of hydraulic retention time (HRT) on metal accumulation was studied. Longer HRTs (>12 h) were associated with greater metal removal than short HRTs, particularly with regard to cadmium and zinc. The system was also shown to operate successfully over an extended period of time, at an HRT of 24 h, with removal efficiencies of approximately 34%, 85% and 57% for Cd²⁺, Cu²⁺ and Zn²⁺ respectively after 5–8 weeks contact. Journal of Industrial Microbiology & Biotechnology (2000) 24, 244–250. Received 28 July 1999/ Accepted in revised form 21 December 1999     

Synergistic or Antagonistic Effect of MTE plus TF or Icariin from <Emphasis Type="Italic">Epimedium koreanum</Emphasis> on the Proliferation and Differentiation of Primary Osteoblasts In Vitro

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test and alkaline phosphatase activity assay were employed to assess the effects of mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ plus total flavonoids or icariin from Epimedium koreanum on the proliferation and differentiation of primary osteoblasts in vitro. The results indicated that icariin (0.1, 1, and 10 μmol/L) and total flavonoids (0.06, 0.6, and 6 μg/mL) inhibited the proliferation and promoted the differentiation of primary osteoblasts. Mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ (0.1, 1, and 10 μmol/L) inhibited the proliferation and promoted the differentiation at 0.1 and 1 μmol/L, but inhibited the differentiation at 10 μmol/L. The effects of mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ plus total flavonoids or icariin from E. koreanum on the proliferation and differentiation of primary osteoblasts in vitro are complicated, and both synergistic and antagonistic effects are generated. The results suggest that there may be a potential cooperative action between flavonoids and trace metal elements on the proliferation and differentiation of primary osteoblasts by forming metal complexes. The combination model between flavonoids and trace metal elements is a pivotal factor for switching the biological effects from toxicity to activity, from damage to protection.     

Effect of extraneous zinc on calf intestinal alkaline phosphatase

The effect of extraneous zinc on calf intestinal alkaline phosphatase was studied for quick reversible binding and slow irreversible binding of zinc ions at various concentrations. Under the conditions of slow binding of zinc to CIP increasing Zn²⁺ (less than 1.0 mM, n_M/n_E 1.0 × 10⁶) inhibited enzymatic activity, and further increasing Zn²⁺ resulted in an increase of activity. For quick reversible binding of Zn²⁺, the effect on CIP activity changed at lower concentrations of substrate, indicating a complex cooperativity between Zn²⁺ and pNPP. Both protein intrinsic emission fluorescence and ANS-bound protein fluorescence, as well as circular dichroism spectra have shown that the binding of zinc ions changed the enzyme conformation, which was the reason for the changes in enzyme activity induced by extraneous zinc.