microM Zinc Research Articles

Zinc and magnesium ions synergistically inhibit superoxide generation by cultured human neutrophils—a promising candidate formulation for amnioinfusion fluid

Oligohydramnios is often caused by the premature rupturing of membranes and subsequent intrauterine infections, such as chorioamnionitis, in which event oxidative stress is hypothesized to be closely associated with the damage to the fetal organs. The clinical efficiency of amnioinfusion using warmed saline in cases of premature rupture of membranes is still controversial, especially concerning the prognosis for the fetus. In the present study, we found that human amniotic fluid per se suppresses the release of superoxide from cultured human neutrophils, suggesting an acute or chronic shortage of amniotic fluid in cases of premature rupture of membranes can affect the shielding of intrauterine organs from oxidative stress. The aim of this study was to propose a formula of zinc and magnesium ions in saline for amnioinfusion, by assessing antioxidative activities. A combination of 5 microM zinc and 5mM magnesium in saline synergistically inhibited superoxide production by cultured human neutrophils, equivalent to human amniotic fluid. The intraperitoneal administration of this formula significantly improved the survival rate in a rat model of peritonitis compared to the saline control (46.7% vs. 10%). The combination of these metals with saline may thus be a promising formula for an amnioinfusion fluid with the capacity to protect fetal organs from oxidative stress.

The Plant Phenolic Diterpene Carnosol Suppresses Sodium Nitroprusside-Induced Toxicity in C6 Glial Cells

Carnosol, a naturally occurring bioactive phenolic diterpene originating from rosemary and sage, has been shown to exert antioxidant and anti-inflammatory effects. This study examined possible protective effects of carnosol on sodium nitroprusside (SNP)-induced cytotoxicity in C6 glial cells. Carnosol (1-10 microM) dose-dependently attenuated SNP (100 microM)-induced cell death and NO production. SNP-induced apoptotic characteristics, including DNA fragmentation, caspase-3 activation, and c-jun N-terminal protein kinase (JNK) phosphorylation, were significantly suppressed by carnosol (10 microM). In addition, carnosol pretreatment restored the level of reduced glutathione (GSH), which was diminished by SNP treatment. Although both SNP (100 microM) and carnosol (10 microM) stimulated the HO-1 expression time-dependently, SNP caused a temporal increase in HO-1 in early time periods (3-6 h) before cell death occurred. In contrast, carnosol induced the sustained expression of HO-1 until a late time point (24 h). The addition of 1 microM zinc protoporphyrin IX (ZnPP), a specific HO inhibitor, with SNP or carnosol further reduced cell viability. Also, the addition of ZnPP inhibited the protective effect of carnosol against SNP-induced cytotoxicity in C6 cells. These results suggest that carnosol possesses abilities to inhibit SNP-mediated glial cell death through modulation of apoptotic events and induction of HO-1 expression.

Neurosteroids differentially modulate P2X4 ATP‐gated channels through non‐genomic interactions

As neuroactive steroids modulate several ionotropic receptors, we assessed whether the ATP-gated currents elicited by P2X(4) receptors are modulated by these compounds. We transfected HEK293 cells or injected Xenopus laevis oocytes with the cDNA coding for rat P2X(4) receptor. Application of 0.1-10 microM alfaxolone potentiated within 60-s the 1 microM ATP-evoked currents with a maximal potentiation of 1.8 and 2.6-fold in HEK293 or oocytes cells respectively. Allopregnalolone or 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (THDOC) also potentiated the ATP-gated currents but with a maximal effect only averaging 1.25 and 1.35-fold respectively. In contrast, 0.3-10 microM pregnanolone, but not its sulfated derivative, inhibited the ATP-gated currents; the maximal inhibition reached 40% in both cell types. THDOC, but not other neurosteroids increased significantly the tau(off) of the ATP-evoked currents, revealing another mode of neurosteroid modulation. Sexual steroids such as 17beta-estradiol or progesterone were inactive revealing explicit structural requirements. Alfaxolone or THDOC at concentrations 30- to 100-fold larger than required to modulate the receptor, gated the P2X(4) receptor eliciting ATP-like currents that were reduced with suramin or brilliant blue G, but potentiated the P2X(4) receptor more than 10-fold by 10 microM zinc. In conclusion, neurosteroids rapidly modulate via non-genomic mechanisms and with nanomolar potencies, the P2X4 receptor interacting likely at distinct modulator sites.

Potassium channels are involved in zinc‐induced apoptosis in MES23.5 cells

There is evidence that zinc may be involved in the pathogenesis of Parkinson's disease by an apoptotic pathway. However, the mechanisms underlying zinc-induced apoptosis are unknown. Previous studies showed that 6-hydroxydopamine (6-OHDA)-enhanced potassium channels are involved in apoptosis of dopaminergic neurons. Our study was designed to test whether zinc-induced apoptosis was mediated by potassium channels. First we demonstrated cell apoptosis with zinc treatment by Hoechst staining assay. The results showed that 13.38% +/- 0.6% of MES23.5 cells were apoptotic after 24 hr of incubation with 60 microM zinc sulfate. Then we observed that the tyrosine hydroxylase (TH) protein expression and the dopamine content decreased, as detected by Western blots and high-performance liquid chromatography-electrochemical detection (HPLC-ECD). We further elucidated the mechanism of cell apoptosis by using whole-cell patch clamp recording. The data demonstrated that MES23.5 cells exhibited a tetraethylammonium (TEA)-sensitive outward K(+) current with delayed rectifier characteristics. Increases of K(+) current density were recorded following the treatment with 60 microM zinc for 4-8 hr. After incubation with 20 mM TEA, the zinc-induced enhancement of K(+) currents was fully blocked. Furthermore, incubation with TEA blocked zinc-mediated caspase-3 activation and cell apoptosis. These data suggest that zinc-induced apoptosis of MES23.5 dopaminergic cells may due to the enhancement of TEA-sensitive K(+) channel activity.

Biochemical alterations in juvenile carp (Cyprinus carpio) exposed to zinc: Glutathione reductase as a target

The aim of this study was to investigate biochemical changes in juvenile carp (Cyprinus carpio) exposed to zinc chloride (10, 30 and 100 microM) for a period of 48 h. Zinc exposure caused a concentration-dependent reduction in glutathione reductase (GR) activity in gills, liver and brain. Gill glutathione S-transferase (GST) was reduced when animals were exposed to the highest concentration of 100 microM zinc. The phosphorylation of p38(MAPK) increased in the brain of fish exposed to zinc 100 microM, while phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and c-Jun N-terminal protein kinase 1/2 (JNK1/2) remained unchanged. Expression of proteins HSP60 and HSP70 were not affected by zinc exposure. Considering the significant concentration-dependent inhibition of GR in all tissues analyzed, this enzyme could be a potential biomarker of exposure to zinc, which has to be confirmed.

Differential gene expression after zinc supplementation and deprivation in human leukocyte subsets.

An individual's zinc status has a significant impact on the immune system, and zinc deficiency, as well as supplementation, modulates immune function. To investigate the effects of zinc on different leukocyte subsets, we used microarray technology to analyze and compare the changes in mRNA expression in cell culture models of monocytes (THP-1), T cells (Jurkat), and B cells (Raji), in response to supplementation for 40 h with 50 microM zinc or 2.5 microM of the membrane-permeant zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine], respectively. In each cell type, several hundred genes were identified to be zinc sensitive, but only a total of seven genes were commonly regulated in all three cell lines. The majority of those genes were involved in zinc homeostasis, and none in immune function. Nevertheless, further analysis revealed that zinc affects entire functional networks of genes that are related to proinflammatory cytokines and cellular survival. Although the zinc-regulated activities are similar throughout the gene networks, the specific genes that are affected vary significantly between different cell types, a situation that helps to elucidate the disparity of the effects that zinc has on different leukocyte populations.

Differential Gene Expression after Zinc Supplementation and Deprivation in Human Leukocyte Subsets

An individual's zinc status has a significant impact on the immune system, and zinc deficiency, as well as supplementation, modulates immune function. To investigate the effects of zinc on different leukocyte subsets, we used microarray technology to analyze and compare the changes in mRNA expression in cell culture models of monocytes (THP-1), T cells (Jurkat), and B cells (Raji), in response to supplementation for 40 h with 50 microM zinc or 2.5 microM of the membrane-permeant zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine], respectively. In each cell type, several hundred genes were identified to be zinc sensitive, but only a total of seven genes were commonly regulated in all three cell lines. The majority of those genes were involved in zinc homeostasis, and none in immune function. Nevertheless, further analysis revealed that zinc affects entire functional networks of genes that are related to proinflammatory cytokines and cellular survival. Although the zinc-regulated activities are similar throughout the gene networks, the specific genes that are affected vary significantly between different cell types, a situation that helps to elucidate the disparity of the effects that zinc has on different leukocyte populations.

Splice Variants of the Human Zinc Transporter ZnT5 (SLC30A5) Are Differentially Localized and Regulated by Zinc through Transcription and mRNA Stability

Maintenance of cellular zinc homeostasis includes regulating the expression of cell membrane zinc transporters. Knowledge about the mechanisms underlying changes in mammalian zinc transporter mRNA abundance is poor. We demonstrated that when expressed in Chinese hamster ovary cells as N-terminal fusions to green fluorescent protein, two splice variants of ZnT5 adopt different subcellular locations (either in the Golgi apparatus or throughout the cell, including at the plasma membrane) indicating discrete roles in cellular zinc homeostasis. We demonstrated, using a beta-galactosidase reporter gene, that both splice variants were expressed from a promoter region that was transcriptionally repressed by increased extracellular zinc (150 microM compared with 3 mum; approximately 40%) and by extracellular zinc depletion, using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine ( approximately 20%). We mapped the zinc-responsive element to the region -154 to +50, relative to the predicted start of transcription, and showed that a consensus metal response element sequence (-410 to -404) was not responsible for these effects. Changes in ZnT5 mRNA abundance in Caco-2 cells at different zinc concentrations were in parallel to the changes in promoter activity ( approximately 40% reduction at 150 microM zinc) but in the presence of actinomycin D, to prevent transcription, we observed a marked stabilization (1.7-2-fold accumulation over 24 h) of ZnT5 mRNA. We conclude that effects of zinc on ZnT5 transcription and mRNA stability act in opposition to balance mRNA abundance for cellular zinc homeostasis. To our knowledge, this is the first report that zinc affects the stability of a transcript with a direct role in cellular zinc homeostasis.

Adduct simplification in the analysis of cyanobacterial toxins by matrix‐assisted laser desorption/ionization mass spectrometry

A novel method for simplifying adduct patterns to improve the detection and identification of peptide toxins using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry is presented. Addition of 200 microM zinc sulfate heptahydrate (ZnSO(4) . 7H(2)O) to samples prior to spotting on the target enhances detection of the protonated molecule while suppressing competing adducts. This produces a highly simplified spectrum with the potential to enhance quantitative analysis, particularly for complex samples. The resulting improvement in total signal strength and reduction in the coefficient of variation (from 31.1% to 5.2% for microcystin-LR) further enhance the potential for sensitive and accurate quantitation. Other potential additives tested, including 18-crown-6 ether, alkali metal salts (lithium chloride, sodium chloride, potassium chloride), and other transition metal salts (silver chloride, silver nitrate, copper(II) nitrate, copper(II) sulfate, zinc acetate), were unable to achieve comparable results. Application of this technique to the analysis of several microcystins, potent peptide hepatotoxins from cyanobacteria, is illustrated.

The β-Adrenergic Receptor Antagonist Metipranolol Blunts Zinc-Induced Photoreceptor and RPE Apoptosis

To determine the effect of zinc on retinal cells at concentrations at which it is known to cause oxidative stress. Furthermore, the effects of metipranolol, known to prevent retinal damage, and of other antiglaucoma drugs were determined on zinc-injured retinal cells. Lipid peroxidation assays were conducted on rat brain and bovine retina-retinal pigment epithelial (RPE) membrane preparations. Immunohistochemistry, immunoblot analysis and the terminal-deoxynucleotidyl transferase dUTP-linked nick-end labeling (TUNEL) procedure determined the effects of zinc with or without trolox or metipranolol on photoreceptor death in situ. The effect of treatments on cultured RPE cells was analyzed using cell viability assays, immunoblot analysis, and the TUNEL procedure. Zinc-induced lipid peroxidation of rat brain and bovine retina-RPE membranes, although the effect of the latter was of a (twofold) greater magnitude. Both effects, however, were similarly attenuated by metipranolol, desacetylmetipranolol, and trolox. Antiglaucoma drugs other than metipranolol had no effect. Intraocular injection of 150 microM zinc and treatment of cultured RPE cells with zinc led to mainly photoreceptor apoptosis and apoptotic death of RPE cells (50% death at 18 microM rising to 10% at 50 microM), respectively. Zinc-induced apoptosis of cultured RPE cells and photoreceptors were attenuated only by metipranolol and trolox. The combined data suggest that oxidative injury to RPE cells and photoreceptors may be caused by elevated levels of zinc in diseases such as age-related macular degeneration (AMD) and that metipranolol may act as an efficacious antioxidant to blunt this process.

Zinc uptake and storage: the role of fundus pigmentation

Age-related macular degeneration (AMD) is associated with lower melanin pigmentation and is more prevalent among the elderly Caucasian population than among Africans. A correlation between light iris colour, fundus pigmentation and the incidence of AMD is reported. Moreover, melanin represents the main storage of zinc in the eye. Zinc enhances antioxidant capacity through its function as a cofactor of important enzymes or by influencing gene expression of regulatory elements in the eye. In this study, we investigated the uptake and storage of zinc in the human choroid/retinal pigment epithelium (RPE) complexes in dependence on the fundus pigmentation as judged by the iris colour. Choroid/RPE complexes of blue and brown human eyes were used. Tissues without any substitution served as controls. Specimens from choroid/RPE complexes were incubated with 100 microM zinc chloride for 24 h. After incubation, pieces of the complexes were stored to investigate the uptake of zinc. The rest of the tissues were kept for 3 and 7 days in culture medium (DMEM) for storage examination. The concentration of zinc was measured by inductively coupled plasma mass spectrometry. After 24 h of zinc treatment the concentration of zinc in the choroid/RPE complexes of blue eyes was not significantly increased. The concentration of zinc in highly pigmented tissues (brown eyes) was increased by the factor 5.1 after 24 h and remained at high levels after 3 days (factor 4.4) and 7 days (factor 2.8). Zinc uptake in the choroid/RPE complex correlates to the iris colour. Alterations of the degree of iris pigmentation result in differences of zinc uptake and storage in the choroids. A potential protective role of zinc may be more prominent in dark- than in light-coloured eyes.

Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice

We describe the durable correction of streptozotocin-induced murine diabetes by in vivo implantation of primary mouse hepatocytes electroporated ex vivo with a human proinsulin cDNA plasmid construct controlled by glucose and zinc regulatory elements. Transfected hepatocytes increased insulin transgene transcription and secretion within 10-20 min of exposure to 25 mM glucose or 60 microM zinc. Insulin release did not occur from secretory granules. Electroporated Rosa26 hepatocytes ( approximately 8 x 10(5) viable cells) were implanted in C57BL/6J diabetic mice in one of three sites: unresected liver, regenerating liver or mesentery. Control diabetic mice were implanted with untransfected hepatocytes. At 30 days after implantation, 8/15 control mice were alive, while 19/19 treated mice were alive. The ratio of body weight on day 30/nadir body weight was significantly higher for all treated groups compared with controls. All eight surviving control mice were hyperglycemic 30 days post-implantation, while 16/19 treated diabetic mice remained normoglycemic. Treated mice had lower mean glucose values (P< or =0.001) without fasting hypoglycemia and better glucose tolerance (P< or =0.0003) than untreated controls. All (6/6) diabetic mice implanted in regenerating liver and 71% (5/7) implanted in unresected liver were alive 77 days after implantation. Engrafted hepatocytes were identified, mainly around central veins, by staining for beta-galactosidase activity and with anti-human insulin antibody.

Mechanism of zinc‐induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3β in SH‐SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.

Zinc protects against arsenic-induced apoptosis in a neuronal cell line, measured by DEVD-caspase activity

Acute and chronic arsenic exposure results in toxicity in humans and causes many neurological and other manifestations. For the first time the present study reports that zinc decreases arsenic-induced apoptosis and also confirms a single report of apoptosis induced by arsenic in a neuronal cell line. Apoptosis measured by DEVD-caspase activity peaked between 10 microM and 20 microM of arsenic trioxide. Higher concentrations of arsenic up to 40 microM caused increasing cell death with diminishing DEVD-caspase activity. The beneficial effect of zinc was proportional to its concentration with a significant decrease in arsenic-induced DEVD-caspase activity at 50 microM and 75 microM zinc (P < 0.05). This finding may be of therapeutic benefit in people suffering from chronic exposure to arsenic from natural sources, a global problem especially relevant to millions of people on the Indian subcontinent.

Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IKSO, by zinc.

Background potassium channels control the resting membrane potential of neurones and regulate their excitability. Two-pore-domain potassium (2-PK) channels have been shown to underlie a number of such neuronal background currents. Currents through human TASK-1, TASK-2 and TASK-3 channels expressed in Xenopus oocytes were inhibited by extracellular acidification. For TASK-3, mutation of histidine 98 to aspartate or alanine considerably reduced this effect of pH. Zinc was found to be a selective blocker of TASK-3 with virtually no effect on TASK-1 or TASK-2. Zinc had an IC(50) of 19.8 microM for TASK-3, at +80 mV, with little voltage dependence associated with this inhibition. TASK-3 H98A had a much reduced sensitivity to zinc suggesting this site is important for zinc block. Surprisingly, TASK-1 also has histidine in position 98 but is insensitive to zinc block. TASK-3 and TASK-1 differ at position 70 with glutamate for TASK-3 and lysine for TASK-1. TASK-3 E70K also had a much reduced sensitivity to zinc while the corresponding reverse mutation in TASK-1, K70E, induced zinc sensitivity. A TASK-3-TASK-1 concatamer channel was comparatively zinc insensitive. For TASK-3, it is concluded that positions E70 and H98 are both critical for zinc block. The native cerebellar granule neurone (CGN) leak current, IK(SO), is sensitive to block by zinc, with current reduced to 0.58 of control values in the presence of 100 microM zinc. This suggests that TASK-3 channels underlie a major component of IK(SO). It has recently been suggested that zinc is released from inhibitory synapses onto CGNs. Therefore it is possible that inhibition of IK(SO) in cerebellar granule cells by synaptically released zinc may have important physiological consequences.

Combined actions of zinc and fluoxetine on nicotinic acetylcholine receptors.

Zinc and nicotinic acetylcholine receptors (nAChRs) seem to be associated with major depression, and some antidepressants, including fluoxetine (Prozac), antagonize nAChRs. Therefore, a study was made of the modulation of neuronal alpha4beta4 and muscle alpha1beta1gammadelta nAChRs, expressing in oocytes, by the combined action of zinc and fluoxetine. At a holding potential of -60 mV, 200 microM zinc increased by 361% the currents elicited by acetylcholine (ACh currents) for alpha4beta4 and by 182% for alpha1beta1gammadelta nAChRs. In contrast, 5 microM fluoxetine reduced the ACh currents to 31% for alpha4beta4 and to 45% for alpha1beta1gammadelta nAChRs. Additionally, fluoxetine reduced more the ACh currents in the presence of zinc: to 17% for alpha4beta4 and to 19% for alpha1beta1gammadelta nAChRs, and after washing out the fluoxetine the ACh current did not recover its zinc-potentiated value. Moreover, when ACh-activated nAChRs were exposed first to fluoxetine and then zinc was added, the potentiating effect of zinc was very small for muscle nAChRs and was nil for neuronal receptors. Thus, the inhibiting effect of fluoxetine prevails over the potentiating action of zinc. Finally, the effects of both zinc and fluoxetine were voltage independent, indicating that these substances interact outside the ion channel. As fluoxetine nullifies the effects of zinc, it appears that both substances interact in the same site. These results should help understand better the roles played by zinc, antidepressants, nAChRs and their combination in brain functions and in the treatment of depression.

The pigmentation of human iris influences the uptake and storing of zinc.

Age-related macular degeneration (AMD) is more prevalent among the elderly Caucasians than in Africans. A significant association between light iris colour, fundus pigmentation and incidence of AMD is reported, suggesting a possible correlation with melanin pigment. Zinc is known to bind to melanin in pigmented tissues and to enhance antioxidant capacity by function as a cofactor or gene expression factor of antioxidant enzymes in the eye. In this in vitro study, we investigated the uptake and storage of zinc in human irides. Irides of blue and brown human eyes were used. The number of melanocytes was measured. Tissues without any treatment served as controls. The irides were incubated with 100 microM zinc chloride in culture medium for 24 h. Specimens of the tissues were stored for the uptake examination. The remained pieces were further incubated for 3 and 7 d to investigate the storage of zinc. The concentration of zinc was measured by inductively coupled plasma mass spectrometry (ICP-MS). Melanocytes count was significantly higher in the brown tissues (P < 0.0001). Zinc concentration of blue coloured irides after 24 h zinc treatment was close to the controls. We did not observe any significant storing. In contrast, the concentration of zinc in brown irides was significantly increased after 24 h (P < or = 0.01) and remained at a high level for 7 d. The uptake of zinc is likely dependent on the amount of pigmentation in human iris. Therefore, we assume that in patients suffering from AMD the degree of pigmentation of the irides and eventually fundi should be under consideration when the patients are treated with zinc supplementation.

Protection by pyruvate of rat retinal cells against zinc toxicity in vitro, and pressure-induced ischemia in vivo.

To examine whether zinc accumulation occurs during retinal neuronal death after pressure-induced ischemia in rats and whether pyruvate protects against such death. To induce transient retinal ischemia, intraocular pressure was increased above systolic pressure for 65 minutes. Pyruvate was administered through the tail vein for 12 hours after ischemia to determine its effect on degeneration of retinal neurons. Retinas were removed and sectioned, and zinc accumulation was visualized with N-(6-methoxy-8-quinolyul)-p-carboxybenzoyl-sylphonamide (TFL-Zn) fluorescence microscopy, and neuronal death was determined with acid fuchsin staining. For in vitro studies, retinal cell cultures were prepared from newborn rat pups and used for experiments at days in vitro (DIV) 7 to 10. After retinal ischemia, staining revealed that most zinc-accumulating neurons were injured neurons, suggesting that endogenous zinc may contribute to ischemic neuronal death in the retina. In vitro studies showed that 15 minutes of exposure to 300 to 500 microM zinc resulted in the death of a substantial number of retinal cells in culture, and that this death was preceded by poly(ADP-ribose) polymerase (PARP)-mediated depletion of nicotinamide-adenine dinucleotide (NAD+) and adenosine triphosphate (ATP). Pyruvate, but not lactate, protected against this zinc-induced cell death in vitro. Consistent with this finding, in vivo studies showed that compared with control rats, pyruvate-treated rats had a substantial reduction in the number of cells showing signs of cell death. The present results suggest endogenous zinc contributes to retinal cell death after ischemia. Pyruvate potently protected against zinc toxicity in cultured rat retinal cells and reduced ischemia-induced cell death in rat retinas.

Differential effects of zinc on functionally distinct human growth hormone mutations.

Human growth hormone (hGH) binds and activates lactogenic receptors by a sequential receptor dimerization mechanism. The affinity for the first lactogenic receptor is increased due to one zinc molecule linking hGH residues H18 and E174, located in helices 1 and 4, respectively, with two adjacent residues in the lactogenic receptor (D187 and H188). Two functionally unique groups of mutant hGHs have been identified. Addition of 25 microM zinc to lactogenic bioassays differentially affects mutant activities based on which group they belong to. One mutation (G120R) is located within the binding surface of hGH that interacts with the second lactogenic receptor. In the presence of endogenous zinc, G120R reduces the maximal activity of hGH without altering either the agonist or antagonist phases of the bell-shaped dose-response curve. Addition of zinc to this assay further reduces the activity of this protein. In contrast, mutations within a hydrophobic motif in hGH that functionally couples the two lactogenic receptor binding sites decrease the sensitivity (right-shift) of the agonist phase of the dose-response curve without similarly affecting the antagonist phase. The addition of zinc to these lactogenic assays increases the sensitivity (left-shifts) of the dose-response curves, largely negating the effect of these mutations. The effects of zinc differentiate between mutations within these two distinct functional motifs by limiting the pool of potential conformations that are available for binding within either of the receptor binding sites of this ligand.

Formulation and characterization of DNA-polyethylenimine-dextran sulfate nanoparticles.

Polyethylenimine (PEI) is a promising non-viral gene delivery polymer that produces high transfection efficiency both in vitro and in vivo. The use of PEI, however, is hindered by its toxicity, reflecting its polycationic nature. In an attempt to decrease this charge-dependent cytotoxicity, a polyanionic polymer, dextran sulfate (DS), has been incorporated into self-assembling PEI-DNA complexes with zinc as stabilizing agent. Spherical particles with a mean particle size of approximately 200 nm and a polydispersity index of 0.2 were achieved using the following optimal conditions: PEI solutions at pH 8, PEI/DS mass ratios of >or=2, and 25 microM zinc sulfate. Plasmid DNA was completely condensed within the nanoparticles as confirmed by an ethidium bromide accessibility assay. This result correlates well with DNase protection studies which find partial protection of the DNA nanoparticles from degradation by the enzyme. The DNA was incorporated into the PEI-DS particles with a high efficiency (>95%) and maintained a primarily supercoiled B-form as determined by gel electrophoresis and circular dichroism. The cytotoxicity of the DNA nanoparticles appeared to decrease as the amount of DS in the formulation was increased and they produced moderate transfection activities that were only modestly inhibited by the presence of serum.