Free Zinc Concentration Research Articles

Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion

The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1h following 3h OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels returned to physiological levels, while cytosolic zinc increased gradually over a 24h time period in viable cells. The increase in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death that occurs with both in vitro and in vivo models of reperfusion.

ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc

In E. coli, ZitB and ZntA are important metal exporters that enhance cell viability under high environmental zinc. To understand their functions in maintaining zinc homeostasis, we applied a novel genetically-encoded fluorescent zinc sensor to monitor the intracellular free zinc changes in wild type, ∆zitB and ∆zntA E. coli cells upon sudden exposure to toxic levels of zinc (“zinc shock”). The intracellular readily exchangeable zinc concentration (or “free” zinc) increases transiently from picomolar to nanomolar levels, accelerating zinc-activated gene transcription. After zinc shock, the zitB mRNA level is constant while the zntA mRNA increases substantially in a zinc-dependent manner. In the ∆zitB E. coli strain the free zinc concentration rises more rapidly after zinc shock compared to wild type cells while a prolonged accumulation of free zinc is observed in the ∆zntA strain. Based on these results, we propose that ZitB functions as a constitutive, first-line defense against toxic zinc influx, while ZntA is up-regulated to efficiently lower the free zinc concentration. Furthermore, the ZntR-mediated transcription of zntA exhibits an apparent K1/2 for zinc activation in the nanomolar range in vivo, significantly higher than the femtomolar affinity for zinc binding and transcription activation previously measured in vitro. A kinetically-controlled transcription model is sufficient to explain the observed regulation of intracellular free zinc concentration by ZntR and ZntA after zinc shock.

Molecular Determinants of the Human Zinc Transporter, Hzip4

Zinc is an essential micronutrient which is required for the function of hundreds of cellular enzymes. In addition, zinc is the second most abundant transition metal found in biological systems (iron is most abundant). However, the concentration of free zinc is nano to picomolar since most zinc is bound to proteins. This makes investigating the mechanism of zinc transport across the plasma membrane a challenge. Our interest has been to elucidate the mechanism of zinc transport mediated by one member of the ZIP family of proteins. To this end, we have developed a radiometric uptake assay to study the mechanism of zinc transport. In addition, we have expressed and purified specific domains of the protein to examine the role of these domains in zinc coordination and transport. Thus, using a mixture of biochemical and biophysical techniques, we have begun to describe the mechanism of zinc transport to gain insight into the function of this protein.

Comparison of intracellular zinc signals in nonadherent lymphocytes from young-adult and elderly donors: role of zinc transporters (Zip family) and proinflammatory cytokines

Intracellular zinc homeostasis is crucial in regulating the inflammatory/immune response at any age. It is tightly regulated by zinc transporters that control influx, efflux and compartmentalization of zinc within the cells. Specific methods for detecting the age-related differences in intracellular zinc signaling are poorly described. We report a novel assay induced after the in vitro zinc addition in peripheral blood mononuclear cells (PBMCs) and in lymphocytes from young and old donors in the absence/presence of in vitro zinc depletion (using EDTA). The intracellular labile zinc variations are monitored over time by flow cytometry using Fluozin-3 AM probe. The best curve fit of the data is calculated using a nonlinear regression model defined as follows: pr3/[1+Exp(−pr1−pr2⁎Xt)]. Pr1 depends on the initial free zinc value (time 0); pr2 describes the rate of the speed in reaching the maximum intracellular free zinc concentration; pr3 represents the maximum intracellular zinc increment (plateau curve); Xt is the time course. Age-related intracellular free zinc variations occur in PBMCs and lymphocytes incubated in EDTA-supplemented medium. The higher plateau of the curve (pr3) was observed in younger subjects. An up-regulation of Zip genes (Zip1, Zip2, Zip3), influencing zinc influx, is more pronounced in the young than old donors. Interleukin-6 and tumor necrosis factor-α overproduction was enhanced in old individuals, suggesting the presence of more marked zinc deficiency and chronic inflammation. In conclusion, the determination of intracellular zinc signals induced by in vitro zinc addition using logistic parameters may be useful to estimate the rate of intracellular zinc homeostasis and its role in inflammatory/immune response in aging.

Dependence of the histofluorescently reactive zinc pool on zinc transporter-3 in the normal brain

In the brain, free zinc levels are under the exquisite control of a variety of zinc-regulating systems, in which zinc transporter (ZnT) proteins play a central role. ZnT3, which is prominently expressed in the brain, facilitates the concentration of free zinc in pre-synaptic vesicles. In addition to histochemical staining methods, a variety of zinc-specific fluorescence dyes has been developed to image or analyze zinc in brain tissue. In this study, we demonstrate the close correlations between histofluorescently reactive zinc and ZnT3. We examined the overlapping distribution of the zinc-specific fluorescent dye, N-(6-methoxy-8-quinolyl)- p-toluenesulfonamide (TSQ)-, and ZnT3-immunoreactive fluorescence throughout the normal brain. TSQ and ZnT3-antibody intensely stained the hippocampus, cortex and amygdala, highlighting the characteristic laminar organization of these regions by variably staining the different layers. TSQ fluorescence and ZnT3 immunoreactivity were roughly co-localized with synaptophysin along the neuropil, but were absent in the neuronal soma. However, albeit relatively faint, TSQ fluorescence was also found throughout the brains of ZnT3-knockout mice. Although these results may indicate the presence of very small cerebral free zinc pools distinct from synaptic vesicle zinc, the synaptic vesicle zinc pool is predominant, accounting for more than 95% of the entire histofluorescently reactive zinc pool in the hippocampus and cortex. Thus, the physiological activity of free zinc in the normal brain might largely depend on the pool of synaptic vesicle zinc that is determined by ZnT3.

Redox biochemistry of mammalian metallothioneins

Metallothionein (MT) is a generic name for certain families of structurally rather variable metal-binding proteins. While purely chemical or biological approaches failed to establish a single physiologic function for MTs in any species, a combination of chemical and biological approaches and recent progress in defining the low but significant concentrations of cytosolic free zinc(II) ions have demonstrated that mammalian MTs function in cellular zinc metabolism in specific ways that differ from conventional knowledge about any other metalloprotein. Their thiolate coordination environments make MTs redox-active zinc proteins that exist in different molecular states depending on the availability of cellular zinc and the redox poise. The zinc affinities of MTs cover a range of physiologic zinc(II) ion concentrations and are modulated. Oxidative conditions make more zinc available, while reductive conditions make less zinc available. MTs move from the cytosol to cellular compartments, are secreted from cells, and are taken up by cells. They provide cellular zinc ions in a chemically available form and participate in cellular metal muffling: the combination of physiologic buffering in the steady state and the cellular redistribution and compartmentalization of transiently elevated zinc(II) ion concentrations in the pre-steady state. Cumulative evidence indicates that MTs primarily have a redox-dependent function in zinc metabolism, rather than a zinc-dependent function in redox metabolism.

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur

Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M- and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes ( znuA, rpmF2 ) more sensitively than others ( rpmG2 , SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5 × 10 −16 M and 7.9 × 10 −16 M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.

Zinc differentially regulates mitogen-activated protein kinases in human T cells

Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.

Imaging Intracellular and Intra-Mitochondrial Free Zinc Ion Concentrations Following Hypoxia/Hypoglycemia with an Expressible Fluorescence Biosensor

Zinc is a “trace” metal necessary for cellular function, but excess free zinc ion is toxic to most cells (1,2,9) We and others have observed increased intra- and extra-cellular free zinc concentrations following cerebral ischemia (3,4). Substantial evidence indicates that mitochondrial dysfunction plays a significant role in neuronal death following ischemia (5), and both mitochondrial dysfunction and increased intracellular zinc have been associated with increased production of reactive oxygen species (ROS) and ultimately apoptosis (6,7). Zinc, potently, inhibits mitochondrial enzymes involved in energy production and destruction of ROS, potentially promoting mitochondrial dysfunction (8). We targeted our expressible fluorescent zinc biosensor (9) to mitochondria of PC12 cells, to ratiometrically image the intra-mitochondrial zinc concentration at resting (pM) levels. We imaged cytoplasmic and mitochondrial zinc levels in cells deprived of oxygen and glucose (OGD), a widely used model of ischemia. Our data indicate that both intra-mitochondrial and cytoplasmic zinc concentrations increase substantially following OGD. Further experiments indicate that the zinc is released from intracellular sites rather than entering the cell from external sources. Supported by NIH EB03924. 1. Canzoniero, L.M., et al. (1999) Journal of Neuroscience 19:RC31, 1–6 2. Zodl, B, et al. (2003) Journal of Inorganic Biochemistry 97, 324–330 3. Tonder, N., et al. (1990) Neuroscience Letters 109, 247–252 4. Frederickson, C.J., et al (2006) Experimental Neurology 198, 285–293 5. Fiskum G., et al. (2008) Mitochondria and Oxidative Stress in Neurodegenerative Disorders: Annals of the New York Academy of Science. 1147, 129–138 6. Weiss, J.H., et al. (2000) Trends in Pharmacological Science 21, 395–4017. Jiang, D., et al. (2001) Journal of Biological Chemistry 276, 47524–47529 8. Gazaryan, I.G., et al. (2007) Journal of Biological Chemistry 282, 24373–24380 9. Bozym, R.A., et al. (2006) ACS Chemical Biology 1, 103–111.

Use of TXRF and Convectional Energy Dispersive X‐ray Fluorescence Analysis (EDXRF) to determine trace metal concentrations in waters of Nakivubo Channel and Lake Victoria

To understand better the pollution levels in the waters of the Nakivubo Channel and Lake Victoria, the concentrations of manganese (Mn), iron (Fe), cobalt (Co), copper (Cu), zinc (Zn) and lead (Pb) were determined using convectional Energy Dispersive X-ray Fluorescence Analysis (EDXRF) and Total X-ray Fluorescence (TXRF) analysis. Particulate deposits were analysed for trace metals with a convectional EDXRF spectrometer. Extracted dissolved metals contents were analysed with Total Reflection X-Ray Fluorescence. The analyses indicated higher copper concentrations in the filtrate samples collected at the rivermouths and inshore stations than on the particulate matter. Samples from battery manufacturing industry-1 indicated copper concentrations in the filtrate exceeding the National Environmental management Authority (NEMA) drinking water standard of 1.0 mg L−1. Free zinc concentrations were measured for almost all the sampling sites, but at concentrations below the 3 mg L−1 NEMA standard. High concentrations of iron in the labile form measured at the lake shores were above NEMA drinking water standards of 0.3–3.5 mg L−1 in 2006, except for the April 2006 Murchison Bay rivermouth, and for low manganese concentrations in the lake waters. The iron and manganese concentrations on the particulate matter at the upstream end of the Channel, but were lower in the lake waters. Effluents from soap manufacturing industries exhibited elevated total iron concentrations, ranging from 19.038 ± 0.190 to 63.129 ± 6.248 mg L−1 throughout the 2-year study period. The manganese concentrations were the highest for the battery manufacturing industry-2 site in April 2006. The total iron and manganese concentrations were generally higher upstream along the Nakivubo Channel than in the lake waters. Cobalt and lead concentrations were below detection limits for most of the sampling sites. Generally, most metal concentrations along the Nakivubo Channel exceeded acceptable limits, illustrating the need for mitigation measures.

Tissue Plasminogen Activator Alters Intracellular Sequestration of Zinc through Interaction with the Transporter ZIP4

Glutamatergic neurons contain free zinc packaged into neurotransmitter-loaded synaptic vesicles. Upon neuronal activation, the vesicular contents are released into the synaptic space, whereby the zinc modulates activity of postsynaptic neurons though interactions with receptors, transporters and exchangers. However, high extracellular concentrations of zinc trigger seizures and are neurotoxic if substantial amounts of zinc reenter the cells via ion channels and accumulate in the cytoplasm. Tissue plasminogen activator (tPA), a secreted serine protease, is also proepileptic and excitotoxic. However, tPA counters zinc toxicity by promoting zinc import back into the neurons in a sequestered form that is nontoxic. Here, we identify the zinc influx transporter, ZIP4, as the pathway through which tPA mediates the zinc uptake. We show that ZIP4 is upregulated after excitotoxin stimulation of the mouse, male and female, hippocampus. ZIP4 physically interacts with tPA, correlating with an increased intracellular zinc influx and lysosomal sequestration. Changes in prosurvival signals support the idea that this sequestration results in neuroprotection. These experiments identify a mechanism via which neurons use tPA to efficiently neutralize the toxic effects of excessive concentrations of free zinc.

Monochloramine-induced toxicity and dysregulation of intracellular Zn2+in parietal cells of rabbit gastric glands

Monochloramine (NH(2)Cl) is a potent, thiol-directed oxidant capable of oxidizing thiol (S-H) residues in a wide variety of proteins. Generated in the stomach by the interaction of bacterial and host products, monochloramine has been shown to dysregulate Ca(2+) homeostasis and disrupt mucosal integrity. In this report, we show that monochloramine also leads to disturbances in intracellular free zinc concentration ([Zn(2+)](i)) in the gastric gland of the rabbit and that the increased Zn(2+) within the cell causes an independent decrease in cell viability. Changes in [Zn(2+)](i) were measured by using the fluorescent reporter FluoZin-3, whereas cell viability was assessed by measuring the conversion of calcein-AM to fluorescent calcein, an assay that is not affected by intracellular oxidation state. Cell death was confirmed using propidium iodide and YO-PRO-1 dye uptake measurements. Our experiments demonstrate that [Zn(2+)](i) is increased in gastric glands exposed to NH(2)Cl and that elevated [Zn(2+)](i) decreases cell viability. Chelation of Zn(2+) with tetrakis-(2-pyridylmethyl) ethylenediamine decreases the toxicity of NH(2)Cl, but only when administered concurrently. These findings suggest that the toxic effect of thiol oxidants present during chronic gastritis is partially due to dysregulation of [Zn(2+)](i) early in the process and that zinc chelation can protect, but not rescue, gastric glands exposed to toxic doses of NH(2)Cl.

Measuring Readily Exchangeable Zinc in Escherichia coli by Carbonic Anhydrase‐based Expressible Ratiometric Zinc Sensor

Zinc is essential in numerous cellular processes and its homeostasis is highly regulated in living organisms. Monitoring readily exchangeable zinc concentration in the cells under various conditions will provide insight into the regulation of zinc homeostasis. Here we engineered a series of genetically encoded fluorescence resonance energy transfer (FRET)‐based excitation ratiometric zinc sensors based on carbonic anhydrase (CA) that exhibit large signal increases in response to zinc. A fluorescent inhibitor of carbonic anhydrase, Dapoxyl sulfonamide (DPS), binds selectively to the active site zinc of CA, undergoing >100 fold increase in quantum yield. A red fluorescent protein (RFP) fused to the C‐terminus of CA functions as the FRET acceptor to zinc‐dependent DPS emission. The FRET signal normalized by the fluorescence from RFP is then calibrated in a series of chelated zinc buffers. These CA_RFP sensors were expressed in the E. coli strain BL21(DE3) in minimal medium and the readily exchangeable zinc concentration was measured at 30 ± 10 pM. The zinc concentration is not dependent on the sensor concentration or the growth stage but varies significantly (from 5 pM to > 300 pM) with changes in the zinc content in the medium. Furthermore, the cells’ ability to maintain low intracellular free zinc concentration is impaired during sugar starvation. This work was supported by National Institutes of Health (NIH) grant NIH/NIBIB RO1 EB003924.

Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes

Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.

Nitric oxide synthase activation and oxidative stress, but not intracellular zinc dyshomeostasis, regulate ultraviolet B light-induced apoptosis

Aims To investigate the role of nitric oxide synthase (NOS) and intracellular free zinc ion (Zn 2+) in regulation of ultraviolet B light (UVB)-induced cell damage and apoptosis. Main methods Real-time confocal microscopy measurement was used to determine the changes of intracellular free zinc concentration under different conditions. Cell apoptotic death was determined using fluorescein isothiocyanate (FITC) conjugated-annexin V (ANX5)/PI labeling followed by flow cytometry. Western analysis was used to determine cell apoptosis and eNOS uncoupling. Key findings UVB induced an elevation of Zn 2+ within 2 min of exposure. The UVB-induced intracellular Zn 2+ elevation was dependent on the increase of constitutive nitric oxide synthase (cNOS) activity and production of superoxide. Removal of Zn 2+ with a lower concentration (< 25 μM) of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn 2+-specific chelator, did not induce cell death or prevent cells from UVB-induced apoptosis. However, a higher [TPEN] (> 50 μM) was cytotoxic to cells, but prevented cells from further UVB-induced apoptosis. The higher [TPEN] also induced cNOS uncoupling. Furthermore, treating the cells with a membrane permeable superoxide dismutase (PEG-SOD) inhibited Zn 2+ release and reduced apoptotic cell death after UVB treatment. The results demonstrated a complex and dynamic regulation of UVB-induced cell damage. Significance Our findings not only advance our understanding of the correlations between cNOS activation and Zn elevation, but also elucidated the role of cNOS in regulation of oxidative stress and apoptosis upon UVB-irradiation.

Heat shock but not cold shock leads to disturbed intracellular zinc homeostasis

The heat shock response is a highly conserved process essential for surviving environmental stress, including extremes of temperature. To investigate whether heat shock has an impact on intracellular Zn(2+) homeostasis, cells were subjected to heat shock, and subsequently the intracellular free zinc concentration was investigated. Sublethal heat shock induced a temperature-dependent and transient intracellular Zn(2+) release that was repeatable after 24 h. The free zinc was localized in round-shaped nuclear bodies identified as nucleoli. Metallothionein, the main cellular zinc storing protein, was found to be not functionally essential for this heat-shock-induced effect. No significant oxidative stress within the cells was detected after heat shock. Cold shock and subsequent rewarming did not result in disturbed intracellular zinc homeostasis. These results show that heat shock and cold shock differ with respect to intracellular Zn(2+) release. A role for zinc as signaling ion during fever is conceivable.

Identification of LIV1, a Putative Zinc Transporter Gene Responsible for HDACi-Induced Apoptosis, Using a Functional Gene Screen Approach

Histone deacetylase inhibitors (HDACi) show promise as a novel class of antitumoral agents and have shown the ability to induce apoptosis of tumor cells. To gain a better understanding of the action of HDACi, we conducted a functional gene screen approach named suppression of mortality by antisense rescue technique to identify the key genes responsible for the tumor-selective killing trichostatin A. Over 20 genes associated with HDACi-induced mortality were identified. One of the confirmed positive hits is LIV1, a putative zinc transporter. LIV1 is significantly induced by treatment with HDACi in a number of tumor cells, but not in normal cells. Knockdown of LIV1 suppressed apoptosis induced by HDACi in tumor cells. Although HDACi induced a slight increase in the free intracellular zinc concentration, knockdown of LIV1 significantly enhanced the intracellular zinc level, which was associated with resistance to apoptosis. On the other hand, pretreatment of the cells with a specific zinc chelator TPEN reversed the apoptosis resistance conferred by knockdown of LIV1. However, the biological effects of TPEN were abolished by addition of physiologic concentrations of zinc. Taken together, the present study identifies LIV1 as a critical mediator responsible for HDACi-induced apoptosis. The effect of LIV1 is, at least in part, mediated by affecting intracellular zinc homeostasis, which may be related to alteration of the catalytic activity of the Caspase 3 and expression of some BCL-2 family genes. As such, these findings highlight a novel mechanism underlying the action of HDACi that could be potentially useful in the clinical setting.

Tracing of intracellular zinc(II) fluorescence flux to monitor cell apoptosis by using FluoZin‐3AM

Changes in the free zinc(II) concentration are closely related to cell proliferation and apoptosis, especially during the early apoptotic process. In the present paper, we demonstrated that zinc(II) probe FluoZin-3AM owns sensitive properties to distinguish different stages of apoptotic cell (induced by an anticancer agent, etoposide) according to trace intracellular zinc(II) fluorescence flux. When apoptosis in HeLa or K562 cells was artificially induced, FluoZin-3AM selectively and strongly stained apoptotic cells only at early and middle stages, which was attributed to significantly increased free zinc(II) flux during these stages. This conclusion was further verified by comparing it with the conventional apoptosis detector probe Annexin-V-FITC and PI. Furthermore, FluoZin-3AM was found cell permeable to detect the intracellular zinc(II) fluorescence enhancement to threefolds within 120 s with low cytotoxicity when zinc(II) was incorporated into the cell by zinc(II) ionophore pyrithione. All the above implied that monitoring intracellular zinc fluorescence flux was an effective method to distinguish cell apoptosis from necrosis, and FluoZin-3AM was found to be a suitable probe acting alone to fulfill the work.

QS389. Disturbances in Free Zinc Concentration and Zinc Binding Capacity of the Plasma in Hemorrhagic Shock

QS389. Disturbances in Free Zinc Concentration and Zinc Binding Capacity of the Plasma in Hemorrhagic Shock

Simulating in vitro transcriptional response of zinc homeostasis system in Escherichia coli

BackgroundThe zinc homeostasis system in Escherichia coli is one of the most intensively studied prokaryotic zinc homeostasis systems. Its underlying regulatory machine consists of repression on zinc influx through ZnuABC by Zur (Zn2+ uptake regulator) and activation on zinc efflux via ZntA by ZntR (a zinc-responsive regulator). Although these transcriptional regulations seem to be well characterized, and there is an abundance of detailed in vitro experimental data available, as yet there is no mathematical model to help interpret these data. To our knowledge, the work described here is the first attempt to use a mathematical model to simulate these regulatory relations and to help explain the in vitro experimental data.ResultsWe develop a unified mathematical model consisting of 14 reactions to simulate the in vitro transcriptional response of the zinc homeostasis system in E. coli. Firstly, we simulate the in vitro Zur-DNA interaction by using two of these reactions, which are expressed as 4 ordinary differential equations (ODEs). By imposing the conservation restraints and solving the relevant steady state equations, we find that the simulated sigmoidal curve matches the corresponding experimental data. Secondly, by numerically solving the ODEs for simulating the Zur and ZntR run-off transcription experiments, and depicting the simulated concentrations of zntA and znuC transcripts as a function of free zinc concentration, we find that the simulated curves fit the corresponding in vitro experimental data. Moreover, we also perform simulations, after taking into consideration the competitive effects of ZntR with the zinc buffer, and depict the simulated concentration of zntA transcripts as a function of the total ZntR concentration, both in the presence and absence of Zn(II). The obtained simulation results are in general agreement with the corresponding experimental data.ConclusionSimulation results show that our model can quantitatively reproduce the results of several of the in vitro experiments conducted by Outten CE and her colleagues. Our model provides a detailed insight into the dynamics of the regulatory system and also provides a general framework for simulating in vitro metal-binding and transcription experiments and interpreting the relevant experimental data.