Reactions of the Zn Proteome with Cd2+ and Other Xenobiotics: Trafficking and Toxicity.

Understanding the molecular basis of inorganic chemical toxicity has lagged behind the proliferation of detailed mechanisms that explain the biochemical toxicology of many organic xenobiotics. In this perspective, general barriers to explicating the bioinorganic chemistry of toxic metals are considered, followed by a detailed examination of these issues in relation to the toxicology of Cd2+. The hypothesis is evaluated that Cd2+ damages cells by replacing Zn2+ in key Zn proteins. An emerging methodology to assess the speciation of metals among cell proteins is described. Then, a more general hypothesis is suggested, namely, that the Zn proteome is also the toxicological target of other metals such as Pb2+ as well as NO and reactive oxygen species. The latter may damage cells by altering the structure and function of Zn2+ binding sites that include thiol ligands. In the process, labilized Zn2+ may also perturb cell biochemistry. Lastly, reactions of metal chelating ligands with the Zn proteome, including formation of ligand-Zn protein adducts, provide other potential avenues of biochemical toxicity.

Proteomic High Affinity Zn2+ Trafficking: Where Does Metallothionein Fit in?

The cellular constitution of Zn-proteins and Zn-dependent signaling depend on the capacity of Zn2+ to find specific binding sites in the face of a plethora of other high affinity ligands. The most prominent of these is metallothionein (MT). It serves as a storage site for Zn2+ under various conditions, and has chemical properties that support a dynamic role for MT in zinc trafficking. Consistent with these characteristics, changing the availability of zinc for cells and tissues causes rapid alteration of zinc bound to MT. Nevertheless, zinc trafficking occurs in metallothionein-null animals and cells, hypothetically making use of proteomic binding sites to mediate the intracellular movements of zinc. Like metallothionein, the proteome contains a large concentration of proteins that strongly coordinate zinc. In this environment, free Zn2+ may be of little significance. Instead, this review sets forth the basis for the hypothesis that components of the proteome and MT jointly provide the platform for zinc trafficking.

Lights, Chemicals, Action: Studying Red Worms' Responses to Environmental Contaminants.

We have developed an experimental module that introduces high school students to guided scientific inquiry. It is designed to incorporate environmental health and ecological concepts into the basic biology or environmental-science content of the high school curriculum. Using the red worm, a familiar live species that is amenable to classroom experimentation, students learn how environmental agents affect the animal's locomotion by altering sensory neuron-muscle interactions and, as a result, influence its distribution in nature. In turn, the results of these experiments have direct application to human-caused environmental disruptions that cause changes in species distribution and indirectly increase the recognition that environmental chemicals affect human health. Students undertake a series of explorations to identify how red worms sense their environment and then apply that knowledge to understand the effects of chemical exposure on locomotor behavior. The activities are designed to generate critical thinking about neuromuscular processes and environmental pollutants that affect them.

Chemical-Biological Properties of Zinc Sensors TSQ and Zinquin: Formation of Sensor-Zn-Protein Adducts versus Zn(Sensor)2 Complexes.

Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and intact cells. In turn, this study further supports the formation of sensor-Zn-protein adducts as the principal observed fluorescent product during experiments employing these two sensors.

Bringing Real Inquiry-Based Science to Diverse Secondary Educational Environments: A Virtual Zebrafish Laboratory to Investigate Environmental Health.

The goal of the University of Wisconsin-Milwaukee WInSTEP SEPA program is to provide valuable and relevant research experiences to students and instructors in diverse secondary educational settings. Introducing an online experience allows the expansion of a proven instructional research program to a national scale and removes many common barriers. These can include lack of access to zebrafish embryos, laboratory equipment, and modern classroom facilities, which often deny disadvantaged and underrepresented students from urban and rural school districts valuable inquiry-based learning opportunities. An online repository of zebrafish embryo imagery was developed in the Carvan laboratory to assess the effects of environmental chemicals. The WInSTEP SEPA program expanded its use as an accessible online tool, complementing the existing classroom experience of our zebrafish module. This virtual laboratory environment contains images of zebrafish embryos grown in the presence of environmental toxicants (ethanol, caffeine, and nicotine), allowing students to collect data on 19 anatomical endpoints and generate significant amounts of data related to developmental toxicology and environmental health. This virtual laboratory offers students and instructors the choice of data sets that differ in the independent variables of chemical concentration and duration of postfertilization exposure. This enables students considerable flexibility in establishing their own experimental design to match the curriculum needs of each instructor.

Zinc trafficking: 1,10-phenanthroline, glutathione, and other metal binding ligands form adducts with proteomic Zn2.

Hypotheses were tested that the proteome of pig kidney LLC-PK1 cells (i) contains Zn-proteins that react with a diversity of native and pharmacologically active metal-binding ligands to form ternary complexes and (ii) includes proteins that bind Zn2+ nonspecifically and together form ternary adducts with a variety of metal-binding agents. The method to observe ternary complex formation with Zn-proteins and proteome•Zn involved preformation of fluorescent TSQ [6-Methoxy-(8-p-toluenesulfonamido)quinoline]-Zn-proteins and/or proteome•Zn-TSQ adducts followed by competitive reaction with selected ligands. The loss of TSQ-dependent fluorescence signaled the replacement of TSQ by the competing ligand in the starting adducts. In vitro, 1,10-phenanthroline competed effectively with TSQ for binding to Zn-proteins in the proteome. The successful competition of 1,10-phenanthroline with TSQ-Zn-proteins was also observed in cells. Similarly, 1,10-phenanthroline was shown to bind to a sizable fraction of Zn2+ associated adventitiously with proteome (proteome•Zn). Other synthetic ligands that bind to Zn-proteins and proteome•Zn include 2,2-bipyridyl, 8-hydroxyquinoline, 2,2'-dicarboxypyridine, and pyrithione. Such results suggest that ligand binding to such sites may play a role in the observed biological effects of these and other metal-binding molecules. Although cysteine does not significantly compete with TSQ, glutathione displaces TSQ from Zn-proteins and proteome•Zn at concentrations well below those found in cells, implying that ternary complex formation involving glutathione may be physiologically significant.

Reaction of metal-binding ligands with the zinc proteome: zinc sensors and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The commonly used Zn(2+) sensors 6-methoxy-8-p-toluenesulfonamidoquinoline (TSQ) and Zinquin have been shown to image zinc proteins as a result of the formation of sensor-zinc-protein ternary adducts not Zn(TSQ)(2) or Zn(Zinquin)(2) complexes. The powerful, cell-permeant chelating agent N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is also used in conjunction with these and other Zn(2+) sensors to validate that the observed fluorescence enhancement seen with the sensors depends on intracellular interaction with Zn(2+). We demonstrated that the kinetics of the reaction of TPEN with cells pretreated with TSQ or Zinquin was not consistent with its reaction with Zn(TSQ)(2) or Zn(Zinquin)(2). Instead, TPEN and other chelating agents extract between 25 and 35% of the Zn(2+) bound to the proteome, including zinc(2+) from zinc metallothionein, and thereby quench some, but not all, of the sensor-zinc-protein fluorescence. Another mechanism in which TPEN exchanges with TSQ or Zinquin to form TPEN-zinc-protein adducts found support in the reactions of TPEN with Zinquin-zinc-alcohol dehydrogenase. TPEN also removed one of the two Zn(2+) ions per monomer from zinc-alcohol dehydrogenase and zinc-alkaline phosphatase, consistent with its ligand substitution reactivity with the zinc proteome.

Zinc trafficking to apo-Zn-proteins 2. Cellular interplay of proteome, metallothionein, and glutathione.

A recent study investigated the impact of glutathione (GSH) on the transfer of zinc (Zn) from proteome to apo-carbonic anhydrase. Here, we probed the requirement of glutathione for zinc trafficking in LLC-PK1 pig kidney epithelial cells. Depletion of GSH by at least 95% left cells viable and able to divide and synthesize Zn-proteins at the control rate over a 48-h period. Loss of GSH stimulated the accumulation of 2.5x the normal concentration of cellular Zn. According to gel filtration chromatography, differential centrifugal filtration, and spectrofluorimetry with TSQ, the extra Zn was distributed between the proteome and metallothionein (MT). To test the functionality of proteome and/or MT as sources of Zn for the constitution of Zn-proteins, GSH-deficient cells were incubated with CaEDTA to isolate them from their normal source of nutrient Zn. Control cells plus CaEDTA stopped dividing; GSH-depleted cells plus CaEDTA continued to divide at ∼40% the rate of GSH deficient cells. Evidently, proteome and/or MT served as a functional source of Zn for generating Zn-proteins. In vitro insertion of Zn bound to proteome into apo-carbonic anhydrase occurred faster at larger concentrations of Zn bound to proteome. These results support the hypothesis that enhanced transport of Zn into cells drives the conversion of apo-Zn-proteins to Zn-proteins by mass action. Similar results were also obtained with human Jurkat T lymphocyte epithelial cells. This study reveals a powerful new model for studying the chemistry of Zn trafficking, including transport processes, involvement of intermediate binding sites, and constitution of Zn-proteins.

Mammalian metallothionein in toxicology, cancer, and cancer chemotherapy.

The present paper centers on mammalian metallothionein 1 and 2 in relationship to cell and tissue injury beginning with its reaction with Cd²âº and then considering its role in the toxicology and chemotherapy of both metals and non-metal electrophiles and oxidants. Intertwined is a consideration of MTs role in tumor cell Zn²âº metabolism. The paper updates and expands on our recent review by Petering et al. (Met Ions Life Sci 5:353-398, 2009).

Reactions of the fluorescent sensor, Zinquin, with the zinc-proteome: adduct formation and ligand substitution.

Zinquin (ZQ) is a commonly used sensor for cellular Zn(2+) status. It has been assumed that it measures accessible Zn(2+) concentrations in the nanomolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn(2+) bound as Zn-proteins. The predominant class of products were ZQ-Zn-protein adducts that were characterized in vivo and in vitro by a fluorescence emission spectrum centered at about 470 nm, by their migration over Sephadex G-75 as protein not low molecular weight species, by the exclusion of reaction with lipid vesicles, and by their large aggregate concentration. In addition, variable, minor formation of Zn(ZQ)(2) with a fluorescence band at about 490 nm was observed in vivo in each case. Because incubation of isolated Zn-proteome with ZQ also generated similar amounts of Zn(ZQ)(2), it was concluded that this species had formed through direct ligand substitution in which ZQ had successfully competed for protein-bound Zn(2+). Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH), and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQ(ACID) (Zinquin acid, (2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate)) able to bind to one Zn(2+) and extract the other in Zn(2)-ADH, whereas it removed one Zn(2+) from Zn(2)-AP and did not bind to the other. Zinquin ethyl ester (ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate); ZQ(EE)) bound to both proteins without sequestering Zn(2+) from either one. In contrast to a closely related sensor, 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ), neither ZQ(ACID) nor ZQ(EE) associated with Zn-carbonic anhydrase. A survey of reactivity of these sensors with partially fractionated Zn-proteome confirmed that ZQ and TSQ bind to distinct, overlapping subsets of the Zn-proteome.

TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline), a common fluorescent sensor for cellular zinc, images zinc proteins.

Zn(2+) is a necessary cofactor for thousands of mammalian proteins. Research has suggested that transient fluxes of cellular Zn(2+) are also involved in processes such as apoptosis. Observations of Zn(2+) trafficking have been collected using Zn(2+) responsive fluorescent dyes. A commonly used Zn(2+) fluorophore is 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ). The chemical species responsible for TSQ's observed fluorescence in resting or activated cells have not been characterized. Parallel fluorescence microscopy and spectrofluorometry of LLC-PK(1) cells incubated with TSQ demonstrated punctate staining that concentrated around the nucleus and was characterized by an emission maximum near 470 nm. Addition of cell permeable Zn-pyrithione resulted in greatly increased, diffuse fluorescence that shifted the emission peak to 490 nm, indicative of the formation of Zn(TSQ)(2). TPEN (N,N,N'N'-tetrakis(-)[2-pyridylmethyl]-ethylenediamine), a cell permeant Zn(2+) chelator, largely quenched TSQ fluorescence returning the residual fluorescence to the 470 nm emission maximum. Gel filtration chromatography of cell supernatant from LLC-PK(1) cells treated with TSQ revealed that TSQ fluorescence (470 nm emission) eluted with the proteome fractions. Similarly, addition of TSQ to proteome prior to chromatography resulted in 470 nm fluorescence emission that was not observed in smaller molecular weight fractions. It is hypothesized that Zn-TSQ fluorescence, blue-shifted from the 490 nm emission maximum of Zn(TSQ)(2), results from ternary complex, TSQ-Zn-protein formation. As an example, Zn-carbonic anhydrase formed a ternary adduct with TSQ characterized by a fluorescence emission maximum of 470 nm and a dissociation constant of 1.55 × 10(-7) M. Quantification of TSQ-Zn-proteome fluorescence indicated that approximately 8% of cellular Zn(2+) was imaged by TSQ. These results were generalized to other cell types and model Zn-proteins.

Zinc trafficking 1. Probing the roles of proteome, metallothionein, and glutathione.

The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that nonspecific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high-affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase, and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione (GSH). In agreement, inclusion of GSH accelerated the reaction in a concentration-dependent manner. The implications of abundant high-affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with GSH in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: possible reaction of cadmium with the zinc finger domain of Sp1.

Cadmium (Cd) exposure causes glucosuria (glucose in the urine). Previously, it was shown that Cd exposure of primary cultures of mouse kidney cells (PMKC) decreased mRNA levels of the glucose transporters, SGLT1 and SGLT2 and that Sp1 from Cd-exposed cells displayed reduced binding to the GC boxes of the mouse SGLT1 promoter in vitro. Here, we identified a GC box upstream of mouse SGLT2 gene. ChIP assays on PMKC revealed that exposure to 5 microM Cd abolished Sp1 binding to SGLT1 GC box while it decreased Sp1 binding to SGLT2 GC sequence by 30% in vivo. The in vitro DNA binding assay, EMSA, demonstrated that binding of Sp1 from Cd (7.5 microM)-treated PMKC to the SGLT2 GC probe was 86% lower than in untreated cells. Sp1 is a zinc finger protein. Compared to PMKC exposed to 5 microM Cd alone, inclusion of 5 microM Zn restored SGLT1 and 2 mRNA levels by 15% and 30%, respectively. Cd (10 microM) decreased the binding of recombinant Sp1 (rhSp1) to SGLT1 and SGLT2 GC probes to 12% and 8% of untreated controls. Cd exerted no effect on GC-bound rhSp1. Co-treatment with Cd and Zn showed that added Zn significantly restored rhSp1 binding to the SGLT1 and SGLT2. Addition of Zn post Cd treatment was not stimulatory. We conclude that Cd can replace Zn in Sp1 DNA binding domain to reduce its binding to GC sites in mouse SGLT1 and SGLT2 promoters.

Zinc finger transcription factor Zn3-Sp1 reactions with Cd2+.

Cadmium is a major environmental pollutant that causes kidney failure including the inability to resorb nutrients such as glucose. In a mouse kidney cell culture model, Cd(2+) inhibits Na(+)-dependent glucose uptake mediated by SGLT transporters. This defect has been traced to the down-regulation of SGLT mRNA synthesis mediated by the zinc-finger transcription factor, Zn(3)-Sp1. Incubation of Cd(2+) with Zn(2+)-Sp1 inhibited its capacity to bind to GC1, its binding site in the SGLT1 promoter. The extent of reaction was reduced as increasing concentrations of Zn(2+) are simultaneously present in the reaction mixture. The results are consistent with a Cd(2+)-Zn(2+) exchange reaction that inactivates the DNA binding function of the protein. The equilibrium constant for this reaction was calculated as 14 +/- 3 and 7 +/- 4 for the reactions measured by the binding to GC1 and an analogous SGLT2 promoter site. Sequential addition of Cd(2+) and Zn(2+) to Zn(3)-Sp1 failed to inhibit the reduction in DNA binding seen with Cd(2+) alone, indicating that substitution of Zn(2+) by Cd(2+) was followed by a second reaction that failed to respond to Zn(2+). Buffers for the DNA binding reaction (electrophoretic mobility shift assay) contain EDTA and Cd-EDTA is active in the same concentration range as Cd(2+). During the standard 15 min incubation, Cd(2+) down-regulates Zn(3)-Sp1 but is inactive against the adduct, Zn(3)-Sp1.GC1. Kinetic studies demonstrated that with 5 muM Cd(2+), Zn(3)-Sp1 was about 75% inactivated in 15 min, whereas, Zn(3)-Sp1.GC1 was slowly dissociated with 50% still remaining after 60 min. In contrast, Zn(3)-Sp1 bound to a cognate consensus site resisted any reaction over 60 min. An adduct of Zn(3)-Sp1.(polydI-dC) was just as reactive with Cd(2+) as Zn(3)-Sp1. Reexamination of the NMR structure of Zn- and Cd-finger peptides related to Sp1 fingers has revealed subtle changes in conformation of the metalbinding site and DNA-binding helix that occur when Cd(2+) is substituted by Zn(2+).

Reactivity of Zn-, Cd-, and apo-metallothionein with nitric oxide compounds: in vitro and cellular comparison.

The reactivity of Zn(7)- and Cd(7)-metallothionein (MT) with S-nitrosopenicillamine (SNAP), S-nitrosoglutathione (GSNO), and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) was investigated to explore the hypothesis that metallothionein is a signficant site of cellular reaction of nitric oxide or NO compounds. Zn(7)-MT reacted with SNAP or GSNO only under aerobic conditions and in the presence of light, which stimulates the decomposition of S-nitrosothiolates to NO. Zn(2+) is released, and protein thiols are modified. DEA/NO, which degrades spontaneously to release NO, also reacted with Zn(7)-MT only when oxygen was present. Anaerobically, DEA/NO reacted with Zn(7)-MT in the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, which converts NO to NO(2). Glutathione competed effectively with Zn(7)-MT for reactive nitrogen oxide species in reaction mixtures. Reaction of Cd(7)-MT with SNAP also required oxygen and light to react. In this case, only a fraction of the Cd(2+) bound to Cd(7)-MT was displaced by SNAP. Apo-metallothionein was much more reactive with SNAP and DEA-NO than Zn(7)- or Cd(7)-MT. TE671 and LLC-PK(1) cell lines were incubated with DEA/NO to examine the role that MT might play in the cellular reactions of this NO donor compound. Incubation of cells with 0-80 microM Zn(2+) for 24 h resulted in progressively increasing concentrations of Zn-unsaturated MT. One hour of cellular exposure to a range of DEA/NO concentrations followed by 24 h of incubation caused no evident acute toxicity at less than 0.45 mM. Preinduction of MT did not alter this response. The effects of DEA/NO on proteomic, metallothionein, and low molecular weight (LMW) thiol pools, including glutathione (GSH), were measured. Substantial fractions of the proteomic and LMW thiol pools underwent reaction with little dislocation of Zn(2+). In addition, one-third of the MT thiol pool reacted without labilizing any of the bound Zn(2+). These results demonstrated that it was free thiols associated with MT that reacted with DEA/NO not those bound to Zn(2+). Moreover, under the conditions of the experiments, DEA/NO reacted with the spectrum of cellular thiols in proportion to their fraction in the cytosol and did not preferentially react with MT sulfhydryl groups.

Mechanism of cadmium ion substitution in mammalian zinc metallothionein and metallothionein alpha domain: kinetic and structural studies.

Cellular metallothionein (MT) protects against Cd(2+) exposure through direct binding of the metal ion. The model reaction between rabbit liver Zn(7)-MT-2 with Cd(2+) was studied with stopped flow kinetics. Four kinetic steps were observable. Comparison of this reaction with an analog utilizing the MT Zn(4)-alpha domain revealed that only the fastest step involved the Zn(3)-beta domain. Each step of the Zn(4)-alpha domain reaction with Cd(2+) displayed hyperbolic dependence of the observed rate constant on Cd(2+) concentration, with the first step comprising 50% of the total reaction and each of the other two, 25%. The two constants extracted from each of these relationships were interpreted as the equilibrium constant for the initial binding of Cd(2+) to the Zn((4-n)),Cd(n)-thiolate cluster (n = 0-3) of the alpha domain and the first order rate constant for the exchange of Cd(2+) for Zn(2+) in the cluster. Activation enthalpies and entropies were determined for each constant. A suite of Zn((4-n)),Cd(n)-thiolate clusters (n = 0-3) was prepared by titration of the Zn(4)-alpha domain with (113)Cd(2+). The products were analyzed by one-dimensional (113)Cd(2+) NMR spectroscopy to define the distribution of (113)Cd(2+) among the four cluster binding sites. Each of these species was also reacted with Cd(2+). The properties of these reactions were similar to those extracted from the reaction of Cd(2+) with the overall domain. Thus, the kinetic results were linked to (113)Cd(2+) occupancy among the cluster metal binding sites. In turn, this linkage permitted the interpretation of the various constants determined for the reaction of Cd(2+) with the Zn(4)-alpha domain in relation to the alpha domain cluster structure.

Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1.

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn(2+) binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink". Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn(2+) chelating agents including N,N,N',N'-(2-pyridylethyl)ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2-3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK(1) cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn(3)-Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn(3)-Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn(3)-Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK(1) cells to TPEN for 24h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn(2+).

Addressing childhood overweight: strategies learned from one Latino community.

The purpose of this study is to understand one Latino community's perspective about childhood overweight within this high-risk ethnic group. Three focus groups, consisting of 12 mothers, 12 fathers, and 8 boys and 4 girls ages 10-12, participated. Transcripts of interviews were coded using N-VIVO and analyzed thematically. Several themes emerge: parents' demanding work schedules, lack of time, transportation issues, opportunities for physical activities, and lack of meal preparation. Participants knew good nutrition and exercise help prevent obesity. Nursing interventions must address multiple challenges with childhood obesity at the family and community levels.

Detection of Zn2+ release in nitric oxide treated cells and proteome: dependence on fluorescent sensor and proteomic sulfhydryl groups.

Nitric oxide (NO) is both an important regulatory molecule in biological systems and a toxic xenobiotic. Its oxidation products react with sulfhydryl groups and either nitrosylate or oxidize them. The aerobic reaction of NO supplied by diethylamine NONOate (DEA-NO) with pig kidney LLC-PK1 cells and Zn-proteins within the isolated proteome was examined with three fluorescent zinc sensors, zinquin (ZQ), TSQ, and FluoZin-3 (FZ-3). Observations of Zn2+ labilization from Zn-proteins depended on the specific sensor used. Upon cellular exposure to DEA-NO, ZQ sequestered about 13% of the proteomic Zn2+ as Zn(ZQ)2 and additional Zn2+ as proteome·Zn-ZQ ternary complexes. TSQ, a sensor structurally related to ZQ with lower affinity for Zn2+, did not form Zn(TSQ)2. Instead, Zn2+ mobilized by DEA-NO was exclusively bound as proteome·Zn-TSQ adducts. Analogous reactions of proteome with ZQ or TSQ in vitro displayed qualitatively similar products. Titration of native proteome with Zn2+ in the presence of ZQ resulted in the sole formation of proteome·Zn-ZQ species. This result suggested that sulfhydryl groups are involved in non-specific proteomic binding of mobile Zn2+ and that the appearance of Zn(ZQ)2 after exposure of cells and proteome to DEA-NO resulted from a reduction in proteomic sulfhydryl ligands, favoring the formation of Zn(ZQ)2 instead of proteome·Zn-ZQ. With the third sensor, FluoZin-3, neither Zn-FZ-3 nor proteome·Zn-FZ-3 was detected during the reaction of proteome with DEA-NO. Instead, it reacted independently with DEA-NO with a modest enhancement of fluorescence.

Apo-metallothionein emerging as a major player in the cellular activities of metallothionein.

Observations of apo-metallothionein (apo-MT) have been made under a variety of physiologic circumstances, including zinc deficiency in cell culture and in rodents, cellular induction of MT by dexamethasone with concurrent Zn deficiency, a variety of tumors under normal Zn conditions, MT induction by Zn and Bi citrate, induction of hepatic MT after tumor cell injection into nude mice, and overexpression of cardiac MT in MT transgenic mice. Experiments demonstrating both the lability of Zn and Cu bound to MT and the cellular stability of apo-MT are described to help rationalize the widespread presence of this metal-depleted species. Finally, comparative in vitro and cellular experiments examined the relative reactivity of Zn- and apo-MT with nitric oxide species, showing that apo-MT is much more reactive chemically and that in cells it may be a principal reactive species within the MT pool.