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The insidious and deadly nature of mercury's organometallic compounds is informed by two large scale poisonings due to industrial mercury pollution that occurred decades ago in Minamata and Niigata, Japan. The present study examined chemical speciation for both mercury and selenium in a historic umbilical cord sample from a child born to a mother who lived near the Agano River in Niigata. The mother had experienced mercury exposure leading to more than 50 ppm mercury measured in her hair and was symptomatic 9 years prior to the birth. We sought to determine the mercury and selenium speciation in the child's cord using Hg Lα1 and Se Kα1 high-energy resolution fluorescence detected X-ray absorption spectroscopy, the chemical speciation of mercury was found to be predominantly organometallic and coordinated to a thiolate. The selenium was found to be primarily in an organic form and at levels higher than those of mercury, with no evidence of mercury-selenium chemical species. Our results are consistent with mercury exposure at Niigata being due to exposure to organometallic mercury species., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Graham George reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Ingrid Pickering reports financial support was provided by Canada Research Chairs. Ingrid Pickering reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Graham George reports financial support was provided by Canada Research Chairs. Graham George reports financial support was provided by New Frontiers in Research Fund. Ingrid Pickering reports financial support was provided by Canada Foundation For Innovation. Gary Myers reports financial support was provided by National Institute of Environmental Health Sciences., (Copyright © 2023. Published by Elsevier B.V.)

The sulfones are a widespread group of organo-sulfur compounds, which contain the sulfonyl SO 2 group attached to two carbons and have a formal sulfur oxidation state of +2. We have examined the sulfur K near-edge X-ray absorption spectroscopy (XAS) of a range of different sulfones and find substantial spectroscopic variability depending upon the nature of the coordination to the sulfonyl group. We have also examined the sulfur Kβ X-ray emission spectroscopy (XES) of selected representative sulfones. Density functional theory simulations show satisfactory reproduction of both absorption and emission spectra while enabling assignment of the various transitions comprising the spectra. The correspondence between observed and simulated spectra shows promise for ab initio prediction of sulfur X-ray absorption and emission spectra of sulfones of any substituent. The absorption spectra and, to a lesser extent, the emission spectra are sensitive to the nature of the organic groups bound to the sulfonyl (SO 2 ) moiety, clearly showing the potential of X-ray spectroscopy as an in situ probe of sulfone chemistry.

Although Alzheimer's disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The "amyloid cascade hypothesis" proposes that the amyloid β (Aβ) peptide is central to disease pathology, which is supported by elevated Aβ levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The "metals hypothesis" proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aβ, and metal ion chelators have been shown to reverse this reaction in vitro . 8-Hydroxyquinoline-based chelators showed early promise as anti-Alzheimer's drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aβ(1-42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered ∼83% of the Cu(II) from Aβ(1-42), whereas PBT2 sequestered only ∼59% of the Cu(II) from Aβ(1-42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aβ(1-42) species in solution, leaving a single Cu(II)Aβ(1-42) species. It follows that the Cu(II) site in this Cu(II)Aβ(1-42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aβ(1-42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aβ(1-42).

Mercury is ubiquitous in the environment, with rising levels due to pollution and climate change being a current global concern. Many mercury compounds are notorious for their toxicity, with the potential of organometallic mercury compounds for devastating effects on the structures and functions of the central nervous system being of particular concern. Chronic exposure of human populations to low levels of methylmercury compounds occurs through consumption of fish and other seafood, although the health consequences, if any, from this exposure remain controversial. We have used high energy resolution fluorescence detected X-ray absorption spectroscopy to determine the speciation of mercury and selenium in human brain tissue. We show that the molecular fate of mercury differs dramatically between individuals who suffered acute organometallic mercury exposure (poisoning) and individuals with chronic low-level exposure from a diet rich in marine fish. For long-term low-level methylmercury exposure from fish consumption, mercury speciation in brain tissue shows methylmercury coordinated to an aliphatic thiolate, resembling the coordination environment observed in marine fish. In marked contrast, for short-term high-level exposure, we observe the presence of biologically less available mercuric selenide deposits, confirmed by X-ray fluorescence imaging, as well as mercury(II)-bis-thiolate complexes, which may be signatures of severe poisoning in humans. These differences between low-level and high-level exposures challenge the relevance of studies involving acute exposure as a proxy for low-level chronic exposure.

Mercury is in some sense an enigmatic element. The element and some of its compounds are a natural part of the biogeochemical cycle; while many of these can be deadly poisons at higher levels, environmental levels in the absence of anthropogenic contributions would generally be below the threshold for concern. However, mercury pollution, particularly from burning fossil fuels such as coal, is providing dramatic and increasing emissions into the environment. Because of this, the environmental chemistry and toxicology of mercury are of growing importance, with the fate of mercury being vitally dependent upon its speciation. X-ray absorption spectroscopy (XAS) provides a powerful tool for in situ chemical speciation, but is severely limited by poor spectroscopic energy resolution. Here, we provide a systematic examination of mercury Lα1 high energy resolution fluorescence detected XAS (HERFD-XAS) as an approach for chemical speciation of mercury, in quantitative comparison with conventional Hg L III -edge XAS. We show that, unlike some lighter elements, chemical shifts in the Lα1 X-ray fluorescence energy can be safely neglected, so that mercury Lα1 HERFD-XAS can be treated simply as a high-resolution version of conventional XAS. We present spectra of a range of mercury compounds that may be relevant to the environmental and life science research and show that density functional theory can produce adequate simulations of the spectra. We discuss strengths and limitations of the method and quantitatively demonstrate improvements both in speciation for complex mixtures and in background rejection for low concentrations.

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456-490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36-39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Selenium is in many ways an enigmatic element. It is essential for health but toxic in excess, with the difference between the two doses being narrower than for any other element. Environmentally, selenium is of concern due to its toxicity. As the rarest of the essential elements, its low levels often provide challenges to the analytical chemist. X-ray absorption spectroscopy (XAS) provides a powerful tool for in situ chemical speciation but is severely limited by poor spectroscopic resolution arising from core-hole lifetime broadening. Here we explore selenium Kα1 high energy resolution fluorescence detected XAS (HERFD-XAS) as a novel approach for chemical speciation of selenium, in comparison with conventional Se K-edge XAS. We present spectra of a range of selenium species relevant to environmental and life science studies, including spectra of seleno-amino acids, which show strong similarities with S K-edge XAS of their sulfur congeners. We discuss strengths and limitations of HERFD-XAS, showing improvements in both speciation performance and low concentration detection. We also develop a simple method to correct fluorescence self-absorption artifacts, which is generally applicable to any HERFD-XAS experiment.

The compounds of mercury can be highly toxic and can interfere with a range of biological processes, although many aspects of the mechanism of toxicity are still obscure or unknown. One especially intriguing property of Hg(II) is its ability to bind DNA directly, making interstrand cross-links between thymine nucleobases in AT-rich sequences. We have used a combination of small molecule X-ray diffraction, X-ray spectroscopies, and computational chemistry to study the interactions of Hg(II) with thymine. We find that the energetically preferred mode of thymine binding in DNA is to the N3 and predict only minor distortions of the DNA structure on binding one Hg(II) to two cross-adjacent thymine nucleotides. The preferred geometry is predicted to be twisted away from coplanar through a torsion angle of between 32 and 43°. Using 1-methylthymine as a model, the bis -thymine coordination of Hg(II) is found to give a highly characteristic X-ray spectroscopic signature that is quite distinct from other previously described biological modes of binding of Hg(II). This work enlarges and deepens our view of significant biological targets of Hg(II) and demonstrates tools that can provide a characteristic signature for the binding of Hg(II) to DNA in more complex matrices including intact cells and tissues, laying the foundation for future studies of mechanisms of mercury toxicity.

Until recently, sulfur was known as a "spectroscopically silent" element because of a paucity of convenient spectroscopic probes suitable for in situ chemical speciation. In recent years the technique of sulfur K-edge X-ray absorption spectroscopy (XAS) has been used extensively in sulfur speciation in a variety of different fields. With an initial focus on reduced forms of organic sulfur, we have explored a complementary X-ray based spectroscopy - sulfur Kβ X-ray emission spectroscopy (XES) - as a potential analytical tool for sulfur speciation in complex samples. We compare and contrast the sensitivity of sulfur Kβ XES with that of sulfur K-edge XAS, and find differing sensitivities for the two techniques. In some cases an approach involving both sulfur K-edge XAS and sulfur Kβ XES may be a powerful combination for deducing sulfur speciation in samples containing complex mixtures.

8-Hydroxyquinolines (8HQs) comprise a family of metal-binding compounds that have been used or tested for use in numerous medicinal applications, including as treatments for bacterial infection, Alzheimer's disease, and cancer. Two key 8HQs, CQ (5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (2-(dimethylamino)methyl-5,7-dichloro-8-hydroxyquinoline), have drawn considerable interest and have been the focus of many studies investigating their in vivo properties. These drugs have been described as copper and zinc ionophores because they do not cause metal depletion, as would be expected for a chelation mechanism, but rather cellular accumulation of these ions. In studies of their anti-cancer properties, CQ has been proposed to elicit toxic intracellular copper accumulation and to trigger apoptotic cancer cell death through several possible pathways. In this study we used synchrotron X-ray fluorescence imaging, in combination with biochemical assays and light microscopy, to investigate 8HQ-induced alterations to metal ion homeostasis, as well as cytotoxicity and cell death. We used the bromine fluorescence from a bromine labelled CQ congener (5,7-dibromo-8-hydroxyquinoline; B2Q) to trace the intracellular localization of B2Q following treatment and found that B2Q crosses the cell membrane. We also found that 8HQ co-treatment with Cu(ii) results in significantly increased intracellular copper and significant cytotoxicity compared with 8HQ treatments alone. PBT2 was found to be more cytotoxic, but a weaker Cu(ii) ionophore than other 8HQs. Moreover, treatment of cells with copper in the presence of CQ or B2Q resulted in copper accumulation in the nuclei, while PBT2-guided copper was distributed near to the cell membrane. These results suggest that PBT2 may be acting through a different mechanism than that of other 8HQs to cause the observed cytotoxicity.

8-Hydroxyquinolines (8HQs) are a family of lipophilic metal ion chelators that have been used in a range of analytical and pharmaceutical applications over the last 100 years. More recently, CQ (clioquinol; 5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) have undergone clinical trials for the treatment of Alzheimer's disease and Huntington's disease. Because CQ and PBT2 appear to redistribute metals into cells, these compounds have been redefined as copper and zinc ionophores. Despite the attention surrounding the clinical trials and the clear link between 8HQs and metals, the fundamental solution chemistry of how these compounds bind divalent metals such as copper and zinc, as well as their mechanism(s) of action in mammalian systems, remains poorly understood. In this study, we used a combination of X-ray absorption spectroscopy (XAS), high-energy resolution fluorescence detected (HERFD) XAS, electron paramagnetic resonance (EPR), and UV-visible absorption spectroscopies to investigate the aqueous solution chemistry of a range of 8HQ derivatives. To circumvent the known solubility issues with 8HQ compounds and their complexes with Cu(II), and to avoid the use of abiological organic solvents, we have devised a surfactant buffer system to investigate these Cu(II) complexes in aqueous solution. Our study comprises the first comprehensive investigation of the Cu(II) complexes formed with many 8HQs of interest in aqueous solution, and it provides the first structural information on some of these complexes. We find that halogen substitutions in 8HQ derivatives appear to have little effect on the Cu(II) coordination environment; 5,7-dihalogenated 8HQ conformers all have a pseudo square planar Cu(II) bound by two quinolin-8-olate anions, in agreement with previous studies. Conversely, substituents in the 2-position of the 8HQ moiety appear to cause significant distortions from the typical square-planar-like coordination of most Cu(II)-bis-8HQ complexes, such that the 8HQ moieties in the Cu(II)-bis-8HQ complex are rotated approximately 30-40° apart in a "propeller-like" arrangement.

Hundreds of millions of people worldwide are exposed to unacceptable levels of carcinogenic inorganic arsenic. Animal models have shown that selenium and arsenic are mutually protective through the formation and elimination of the seleno-bis(S-glutathionyl) arsinium ion [(GS) 2 AsSe] - . Consistent with this, human selenium deficiency in arsenic-endemic regions is associated with arsenic-induced disease, leading to the initiation of human selenium supplementation trials. In contrast to the protective effect observed in vivo, in vitro studies have suggested that selenite increases arsenite cellular retention and toxicity. This difference might be explained by the rapid conversion of selenite to selenide in vivo. In the current study, selenite did not protect the human hepatoma (HepG2) cell line against the toxicity of arsenite at equimolar concentrations, however selenide increased the IC 50 by 2.3-fold. Cytotoxicity assays of arsenite + selenite and arsenite + selenide at different molar ratios revealed higher overall mutual antagonism of arsenite + selenide toxicity than arsenite + selenite. Despite this protective effect, in comparison to 75 Se-selenite, HepG2 cells in suspension were at least 3-fold more efficient at accumulating selenium from reduced 75 Se-selenide, and its accumulation was further increased by arsenite. X-ray fluorescence imaging of HepG2 cells also showed that arsenic accumulation, in the presence of selenide, was higher than in the presence of selenite. These results are consistent with a greater intracellular availability of selenide relative to selenite for protection against arsenite, and the formation and retention of a less toxic product, possibly [(GS) 2 AsSe] - ., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Organic sulfoxides, a group of compounds containing the sulfinyl S[double bond, length as m-dash]O group, are widespread in nature, important in health and disease, and used in a variety of applications in the pharmaceutical industry. We have examined the sulfur K-edge X-ray absorption near-edge spectra of a range of different sulfoxides and find that their spectra are remarkably similar. Spectra show an intense absorption peak that is comprised of two transitions; a S 1s → (S-O)σ* and a S 1s → [(S-O)π* + (S-C)σ*] transition. In most cases these are sufficiently close in energy that they are not properly resolved; however for dimethylsulfoxide the separation between these transitions increases in aqueous solution due to hydrogen bonding to the sulfinyl oxygen. We also examined tetrahydrothiophene sulfoxide using both the sulfur and oxygen K-edge. This compound has a mild degree of ring strain at the sulfur atom, which changes the energies of the two transitions so that the S 1s → [(S-O)π* + (S-C)σ*] is below the S 1s → (S-O)σ*. A comparison of the oxygen K-edge X-ray absorption near-edge spectra of tetrahydrothiophene sulfoxide with that of an unhindered sulfoxide shows little change, indicating that the electronic environment of oxygen is very similar., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Industrial release of mercury into the local Minamata environment with consequent poisoning of local communities through contaminated fish and shellfish consumption is considered the classic case of environmental mercury poisoning. However, the mercury species in the factory effluent has proved controversial, originally suggested as inorganic, and more recently as methylmercury species. We used newly available methods to re-examine the cerebellum of historic Cat 717, which was fed factory effluent mixed with food to confirm the source. Synchrotron high-energy-resolution fluorescence detection-X-ray absorption spectroscopy revealed sulfur-bound organometallic mercury with a minor β-HgS phase. Density functional theory indicated energetic preference for α-mercuri-acetaldehyde as a waste product of aldehyde production. The consequences of this alternative species in the "classic" mercury poisoning should be re-evaluated.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

Antigen specificity is a primary goal in developing curative therapies for autoimmune disease. Dendritic cells (DCs), as the most effective antigen presenting cells in the body, represent a key target to mediate restoration of antigen-specific immune regulation. Here, we describe an injectable, dual-sized microparticle (MP) approach that employs phagocytosable ∼1 μm and nonphagocytosable ∼30 μm MPs to deliver tolerance-promoting factors both intracellularly and extracellularly, as well as the type 1 diabetes autoantigen, insulin, to DCs for reprogramming of immune responses and remediation of autoimmunity. This poly(lactic- co -glycolic acid) (PLGA) MP system prevented diabetes onset in 60% of nonobese diabetic (NOD) mice when administered subcutaneously in 8 week old mice. Prevention of disease was dependent upon antigen inclusion and required encapsulation of factors in MPs. Moreover, administration of this "suppressive-vaccine" boosted pancreatic lymph node and splenic regulatory T cells (Tregs), upregulated PD-1 on CD4 + and CD8 + T cells, and reversed hyperglycemia for up to 100 days in recent-onset NOD mice. Our results demonstrate that a MP-based platform can reeducate the immune system in an antigen-specific manner, augment immunomodulation compared to soluble administration of drugs, and provide a promising alternative to systemic immunosuppression for autoimmunity., Competing Interests: The authors declare the following competing financial interest(s): J.S.L. and G.P.M. conducted a portion of the reported experiments while employed by OneVax, LLC. C.H.W., T.M.B., M.A.A., and B.G.K. are cofounders of OneVax, LLC, a preclinical biotechnology company with interest in this technology. The authors declare that no other relevant conflicts of interest exist.

Alzheimer's disease (AD) is the main cause of age-related dementia and currently affects approximately 5.7 million Americans. Major brain changes associated with AD pathology include accumulation of amyloid beta (Aβ) protein fragments and formation of extracellular amyloid plaques. Redox-active metals mediate oligomerization of Aβ, and the resultant metal-bound oligomers have been implicated in the putative formation of harmful, reactive species that could contribute to observed oxidative damage. In isolated plaque cores, Cu(II) is bound to Aβ via histidine residues. Despite numerous structural studies of Cu(II) binding to synthetic Aβ in vitro, there is still uncertainty surrounding Cu(II) coordination in Aβ. In this study, we used X-ray absorption spectroscopy (XAS) and high energy resolution fluorescence detected (HERFD) XAS to investigate Cu(II) coordination in Aβ(1-42) under various solution conditions. We found that the average coordination environment in Cu(II)Aβ(1-42) is sensitive to X-ray photoreduction, changes in buffer composition, peptide concentration, and solution pH. Fitting of the extended X-ray absorption fine structure (EXAFS) suggests Cu(II) is bound in a mixture of coordination environments in monomeric Aβ(1-42) under all conditions studied. However, it was evident that on average only a single histidine residue coordinates Cu(II) in monomeric Aβ(1-42) at pH 6.1, in addition to 3 other oxygen or nitrogen ligands. Cu(II) coordination in Aβ(1-42) at pH 7.4 is similarly 4-coordinate with oxygen and nitrogen ligands, although an average of 2 histidine residues appear to coordinate at this pH. At pH 9.0, the average Cu(II) coordination environment in Aβ(1-42) appears to be 5-coordinate with oxygen and nitrogen ligands, including two histidine residues.

Aryl and mixed aryl-alkyl organic sulfides are important species in a variety of fields, including the drug and food industries. They also are present in fossil fuels, where they contribute to the range of sulfur compounds that must be removed by the fuel industry. We have used sulfur K-edge X-ray absorption spectroscopy, in combination with density functional theory calculations, to study the aryl sulfide diphenyl sulfide and two different aryl-alkyl sulfides. The sulfur K near-edge X-ray absorption spectra are strongly affected by the coordination of the phenyl ring and are distinct from spectra of the alkyl sulfides. For diphenyl sulfide the spectra are predicted to be sensitive to rotation about the S-C bonds, with experimental spectra corresponding to a sum of thermally accessible conformations. We also have investigated the vapor-phase spectrum of diphenyl sulfide, which is found to be very similar to that of toluene solutions of the compound.

Mercury is one of the most toxic elements threatening the biosphere, with levels steadily rising due to both natural and human activities. Selenium is an essential micronutrient, required for normal development and functioning of many organisms. While selenium is known to counteract mercury's toxicity under some conditions, to date information about the mercury-selenium relationship is fragmented and often controversial. As part of a systematic study of mercury and selenium interactions, zebrafish (Danio rerio) larvae (a model verterbrate) were exposed to methylmercury chloride or mercuric chloride. The influence of pre- and post-treatment of selenomethionine on the level and distribution of mercury and selenium in the brain and eye sections, as well as on toxicity, were examined. Selenomethionine treatment decreased the amount of maternally transfered mercury in the larval brain. Selenomethionine treatment prior to exposure to mercuric chloride increased both mercury and selenium levels in the brain but decreased their toxic effects. Conversely, methylmercury levels were not changed as a result of selenium pre-treatment, while toxicity was increased. Strikingly, both forms of mercury severely disrupted selenium metabolism, not only by depleting selenium levels due to formation of Hg-Se complexes, but also by blocking selenium transport into and out of tissues, suggesting that restoring normal selenium levels by treating the organism with selenium after mercury exposure may not be possible. Disruption of selenium metabolism by mercury may lead to disruption in function of selenoproteins. Indeed, the production of thyroid hormones by selenoprotein deiodinases was found to be severely impaired as a result of mercury exposure, with selenomethionine not always being a suitable source of selenium to restore thyroid hormone levels.

Background: Selenium is an essential element with a rich and varied chemistry in living organisms. It plays a variety of important roles ranging from being essential in enzymes that are critical for redox homeostasis to acting as a deterrent for herbivory in hyperaccumulating plants. Despite its importance there are many open questions, especially related to its chemistry in situ within living organisms., Scope of Review: This review discusses X-ray spectroscopy and imaging of selenium in biological samples, with an emphasis on the methods, and in particular the techniques of X-ray absorption spectroscopy (XAS) and X-ray fluorescence imaging (XFI). We discuss the experimental methods and capabilities of XAS and XFI, and review their advantages and their limitations. A perspective on future possibilities and next-generation of experiments is also provided., Major Conclusions: XAS and XFI provide powerful probes of selenium chemistry, together with unique in situ capabilities. The opportunities and capabilities of the next generation of advanced X-ray spectroscopy experiments are particularly exciting., General Significance: XAS and XFI provide versatile tools that are generally applicable to any element with a convenient X-ray absorption edge, suitable for investigating complex systems essentially without pre-treatment., (Copyright © 2018. Published by Elsevier B.V.)

Selenium-based selenyl free radicals are chemical entities that may be involved in a range of biochemical processes. We report the first X-ray spectroscopic observation of a selenyl radical species generated photochemically by X-ray irradiation of low-temperature solutions of l-selenocysteine. We have employed high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) and electron paramagnetic resonance (EPR) spectroscopy, coupled with density functional theory calculations, to characterize and understand the species. The HERFD-XAS spectrum of the selenyl radical is distinguished by a uniquely low-energy transition with a peak energy at 12 659.0 eV, which corresponds to a 1s → 4p transition to the singly occupied molecular orbital of the free radical. The EPR spectrum shows the broad features and highly anisotropic g-values that are expected for a selenium free radical species. The availability of spectroscopic probes for selenyl radicals may assist in understanding why life chooses selenium over sulfur in selected biochemical processes.

The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo -Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Copper is an essential nutrient required for many biological processes involved in primary metabolism, but free copper is toxic due to its ability to catalyze formation of free radicals. To prevent toxic effects, in the cell copper is bound to proteins and low molecular weight compounds, such as glutathione, at all times. The widely used chemotherapy agent cisplatin is known to bind to copper-transporting proteins, including copper chaperone Atox1. Cisplatin interactions with Atox1 and other copper transporters are linked to cancer resistance to platinum-based chemotherapy. Here we analyze the binding of copper and cisplatin to Atox1 in the presence of glutathione under redox conditions that mimic intracellular environment. We show that copper(I) and glutathione form large polymers with a molecular mass of approximately 8 kDa, which can transfer copper to Atox1. Cisplatin also can form polymers with glutathione, albeit at a slower rate. Analysis of simultaneous binding of copper and cisplatin to Atox1 under physiological conditions shows that both metals are bound to the protein through copper-sulfur-platinum bridges.

Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017., (© 2017 International Union of Biochemistry and Molecular Biology.)

Aseptic loosening due to peri-prosthetic osteolysis is one of the primary causes for failure of artificial joint replacements. Implant-derived wear particles, often ultra-high molecular weight polyethylene (UHMWPE) microparticles, initiate an inflammatory cascade upon phagocytosis by macrophages, which leads to osteoclast recruitment and activation, ultimately resulting in osteolysis. Investigation into integrin receptors, involved in cellular interactions with biomaterial-adsorbed adhesive proteins, is of interest to understand and modulate inflammatory processes. In this work, we investigate the role of macrophage integrins Mac-1 and RGD-binding integrins in response to UHMWPE wear particles. Using integrin knockout mice as well as integrin blocking techniques, reduction in macrophage phagocytosis and inflammatory cytokine secretion is demonstrated when these receptors are either absent or blocked. Along this line, various opsonizing proteins are shown to differentially modulate microparticle uptake and macrophage secretion of inflammatory cytokines. Furthermore, using a calvarial osteolysis model it is demonstrated that both Mac-1 integrin and RGD-binding integrins modulate the particle induced osteolysis response to UHMWPE microparticles, with a 40% decrease in the area of osteolysis by the absence or blocking of these integrins, in vivo. Altogether, these findings indicate Mac-1 and RGD-binding integrins are involved in macrophage-directed inflammatory responses to UHMWPE and may serve as therapeutic targets to mitigate wear particle induced peri-prosthetic osteolysis for improved performance of implanted joints., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cisplatin remains the most widely used platinum-based anti-cancer drug and is included on the World Health Organization's list of essential medicines. Cisplatin also exhibits severe toxic side-effects, in particular damage to both the kidney and the inner ear, which are thought to derive primarily from hydrolysis products that are more toxic than cisplatin itself. Selective inactivation of these hydrolysis products has emerged as a feasible strategy to mitigate side effects and transform cisplatin into a better medicinal drug. Sodium thiosulfate is one of the most promising of currently considered mitigation agents, and co-administration of large quantities with cisplatin has been shown to considerably reduce toxic side effects in animals without abolishing useful anti-cancer cytotoxicity. The structural basis of this antagonism has, however, remained uncertain. We report herein the structural characterization of the reaction product of hydrolyzed cisplatin and thiosulfate in aqueous solution using X-ray absorption spectroscopy. This reveals the formation of the four-coordinate Pt(II) species [Pt(S 2 O 3 ) 4 ] 6- with Pt-S bond lengths of 2.30Å. Our structural conclusions are supported by density functional theory calculations. More generally speaking, the structural characterization of this Pt-thiosulfate complex reinvigorates the principle strategy to reduce the toxicity of cisplatin (and possibly other platinum-based anticancer drugs) by co-administering appropriate ameliorating agents for direct benefits to patients., (Copyright © 2016. Published by Elsevier Inc.)

Selenium is an essential micronutrient for many organisms, and in vertebrates has a variety of roles associated with protection from reactive oxygen species. Over the past two decades there have been conflicting reports upon human health benefits and detriments arising from consumption of selenium dietary supplements. Thus, early studies report a decrease in the incidence of certain types of cancer, whereas subsequent studies did not observe any anti-cancer effect, and adverse effects such as increased risks for type 2 diabetes have been reported. A possible contributing factor may be that different chemical forms of selenium were used in different studies. Using larval stage zebrafish (Danio rerio) as a model organism, we report a comparison of the toxicities and tissue selenium distributions of four different chemical forms of selenium. We find that the organic forms of selenium tested (Se-methyl-l-selenocysteine and l-selenomethionine) show considerably more toxicity than inorganic forms (selenite and selenate), and that this appears to be correlated with the level of bioaccumulation. Despite differences in concentrations, the tissue specific pattern of selenium accumulation was similar for the chemical forms tested; selenium was found to be highly concentrated in pigment (melanin) containing tissues especially for the organic selenium treatments, with lower concentrations in eye lens, yolk sac and heart. These results suggest that pigmented tissues might serve as a storage reservoir for selenium.

We developed a novel poly(lactic-co-glycolic acid)-based, microparticle (MP) system providing concurrent delivery of multiple encapsulated immuno-suppressive factors and antigen, for in vivo conditioning of dendritic cells (DCs) toward a tolerance promoting pathway. Subcutaneous administration prevents onset of type 1 diabetes (T1D) in NOD mice. Two MP sizes were made: phagocytosable MPs were fabricated encapsulating vitamin D3 or insulin B(9-23) peptide, while unphagocytosable MPs were fabricated encapsulating TGF-β1 or GM-CSF. The combination of Vit D3/TGF-β1 MPs confers an immature and LPS activation-resistant phenotype to DCs, and MP-delivered antigen is efficiently and functionally presented. Notably, two subcutaneous injections into 4week old NOD mice using the combination of MPs encapsulating Vit D3, Ins B, TGF-β1 and GM-CSF protected 40% of mice from T1D development, significant in comparison to the control. This work represents one of the first applications of a biomaterial-based, MP vaccine system to successfully prevent autoimmune diabetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The study of HIV-infected patients using new five-color panel CytoDiff for flow cytofluorometry revealed the imbalance in subpopulations of leukocytes. The manifestations consisted in significant decrease of total amount of lymphocytes and also CD16+ lymphocytes. B-lymphocytes and eosinocytes with synchronous increase of level of CD16+ monocytes, neutrophils and immature B-lymphocytes. After application to patients of highly active anti-retroviral therapy occurred decrease of viral load under synchronous increase of total level of lymphocytes, CD16+ lymphocytes, B-lymphocytes and also decrease of amount of CD16+ monocytes, neutrophils and immature B-lymphocytes.

Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Dendritic cells (DCs), key regulators of tolerance and immunity, have been found to reside in mechanically active tissues such as the interior layers of the arterial wall, which experience cyclic radial wall strain due to pulsatile blood flow. Although experimentally difficult to determine in vivo, it is reasonable to postulate DCs experience the mechanical forces in such mechanically active tissues. However, it is currently unknown how DCs respond to cyclic mechanical strain. In order to explore the hypothesis that DCs are responsive to mechanical strain, DCs were cultured in vitro on pre-adsorbed adhesive proteins (e.g., laminin, collagen, fibrinogen) and 1 Hz cyclic strain was applied for various durations and strain magnitudes. It was determined that a strain magnitude of 10% and 24 h duration adversely affected DC viability compared to no-strain controls, but culture on certain adhesive substrates provided modest protection of viability under this harsh strain regime. In contrast, application of 1 h of 1 Hz cyclic 3% strain did not affect DC viability and this strain regime was used for the remaining experiments for quantifying DC activation and T-cell priming capability. Application of 3% strain increased expression of stimulatory (MHC-II) and costimulatory molecules (CD86, CD40), and this effect was generally increased by culture on pre-coated adhesive substrates. Interestingly, the cytokine secretion profile of DCs was not significantly affected by strain. Lastly, strained DCs demonstrated increased stimulation of allogeneic T-cell proliferation, in a manner that was independent of the adhesive substrate. These observations indicate generation of a DC consistent with what has been described as a semi-mature phenotype. This work begins elucidating a potential role for DCs in tissue environments exposed to cyclic mechanical forces., (Published by Elsevier Ltd.)

Human copper transporters ATP7B (Wilson's disease protein) and ATP7A (Menkes' disease protein) have been implicated in tumour resistance to cisplatin, a widely used anticancer drug. Cisplatin binds to the copper-binding sites in the N-terminal domain of ATP7B, and this binding may be an essential step of cisplatin detoxification involving copper ATPases. In the present study, we demonstrate that cisplatin and a related platinum drug carboplatin produce the same adduct following reaction with MBD2 [metal-binding domain (repeat) 2], where platinum is bound to the side chains of the cysteine residues in the CxxC copper-binding motif. This suggests the same mechanism for detoxification of both drugs by ATP7B. Platinum can also be transferred to MBD2 from copper chaperone Atox1, which was shown previously to bind cisplatin. Binding of the free cisplatin and reaction with the cisplatin-loaded Atox1 produce the same protein-bound platinum intermediate. Transfer of platinum along the copper-transport pathways in the cell may serve as a mechanism of drug delivery to its target in the cell nucleus, and explain tumour-cell resistance to cisplatin associated with the overexpression of copper transporters ATP7B and ATP7A.

Antibacterial coating approaches are being investigated to modify implants to reduce bacterial adhesion and viability in order to reduce implant-associated infection. Nanostructured materials possess unique surface properties, and nanotopographic surfaces have been reported to modulate bacterial adhesion. Zinc oxide (ZnO) films presenting well-controlled nanorod surface structures have recently been developed. To assess the efficacy of ZnO nanorod surfaces as an anti-bacterial coating, we evaluated bacterial adhesion and viability, compared to sputtered ZnO substrates (a relatively flat control) and glass substrates (as a reference). Common implant-associated pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis were investigated. The number of adherent P. aeruginosa on ZnO nanorod surfaces was found to be reduced compared to glass and sputtered ZnO, while the adherent number of S. epidermidis on the ZnO nanorods was equivalent to glass. Regarding bacteria viability, the ZnO nanorod and sputtered ZnO surfaces demonstrated a modest, but significant bactericidal effect on adherent P. aeruginosa, killing 2.5-fold and 1.7-fold more over the number of dead P. aeruginosa on glass, respectively. A greater bactericidal effect of ZnO substrates on S. epidermidis was found, with sputtered ZnO and ZnO nanorod substrates killing -20-fold and 30-fold more over the number of dead S. epidermidis on glass, respectively. These data support the further investigation and optimization of ZnO nanorod coatings with potential for bacterial adhesion resistance and bactericidal properties.

It is known that adsorbed adhesive proteins on implanted biomaterials modulate inflammatory responses; however, modulation of dendritic cell (DC) responses upon interaction with adhesive proteins has only begun to be characterized. DCs are specialized antigen-presenting cells that modulate both innate and adaptive immune responses. Previously we have shown that the activation and stimulatory capacity of DCs derived from C57BL6/j mice is differentially modulated by adhesive substrates. Here we extend our investigation of adhesive substrate modulation of DC responses to consider the case where the DCs had maturational defects associated with diabetes. Understanding the adhesive responses of DCs in diabetics is potentially important for immunotherapy and tissue engineering applications. In this work we use the non-obese diabetic (NOD) mouse, an established animal model for type 1 diabetes, to generate DCs (NOD-DCs). We demonstrate that NOD-DCs cultured on different adhesive substrates (collagen, fibrinogen, fibronectin, laminin, vitronectin, albumin and serum) respond with substrate-dependent modulation of the surface expression of the stimulatory molecule MHC-II and the co-stimulatory molecules CD80 and CD86 and production of the cytokines IL-12p40 and IL-10. Furthermore, the capacity of NOD-DCs to stimulate CD4(+) T-cell proliferation and cytokine production (IL-4 and IFN-γ) showed substrate-dependent modulation. Specifically, NOD-DCs cultured on vitronectin induced the highest IL-12p40 production, whereas collagen induced the highest IL-10 production. Dendritic cells cultured on collagen, fibrinogen and serum-coated substrates stimulated the highest CD4(+) T-cell proliferation. It was further determined that DCs cultured on vitronectin induced the highest percent population of IL-4-producing T-cells and DCs cultured on a fibronectin-coated substrate induced the highest expression of IFN-γ in T-cells. Pearson's correlation analysis revealed high correlations between T-cell proliferation and DC expression level of CD80 and T-cell production of IL-4 and DC production of IL-10. This demonstration of substrate-based control of NOD-DC activatory and stimulatory capacity, distinct from non-diabetic B6-DC responses, establishes the field of adhesive modulation of immune cell responses and informs the rational design of biomaterials for patients with type 1 diabetes., (Published by Elsevier Ltd.)

Dendritic cells (DCs) are central regulators of the immune system that operate in both innate and adaptive branches of immunity. Activation of DC by numerous factors, such as danger signals, has been well established. However, modulation of DC functions through adhesion-based cues has only begun to be characterized. In this work, DCs were cultured on surfaces presenting a uniform gradient of the integrin-targeting RGD peptide generated using the recently established "universal gradient substrate for click biofunctionalization" methodology. Surface expression of activation markers (costimulatory molecule CD86 and stimulatory molecule MHC-II) and production of cytokines IL-10 and IL-12p40 of adherent DCs was quantified in situ. Additionally, bound alpha(V) integrin was quantified in situ using a biochemical crosslinking/extraction method. Our findings demonstrate that DCs upregulated CD86, MHC-II, IL-10, IL-12p40 and alpha(V) integrin binding as a function of RGD surface density, with production of IL-12p40 being the marker most sensitive to RGD surface density. Surface expression of activation markers demonstrated moderate correlation with alpha(V) integrin binding, while cytokine production was highly correlated with alpha(V) integrin binding. This work demonstrates the utility of the surface density gradient platform as a high-throughput method to investigate RGD density-dependent DC adhesive responses. Furthermore, this quantitative analysis of DC integrin-based activation represents a first of its type, helping to establish the field of adhesion-based modulation of DCs as a general mechanism that has previously not been defined, and informs the rational design of biomimetic biomaterials for immunomodulation., (Published by Elsevier Ltd.)

Macrophages associated with implanted biomaterials are primary mediators of chronic inflammation and foreign body reaction to the implant. Hence, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate inflammatory responses. Nanostructured materials possess unique surface properties, and nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. Additionally adherent macrophage number on ZnO flat substrate was reduced as compared to glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. Cell death was apparently not apoptotic, given the lack of activated caspase-3 immunostaining. A decrease in viable macrophage numbers when ZnO substrates were present in the same media verified the role of ZnO substrate dissolution, and dissolved levels of Zn in culture media were quantified. In order to determine long-term physiological responses, ZnO nanorod-coated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 d. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. In conclusion, although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent., (Published by Elsevier Ltd.)

Wilson disease ATPase (ATP7B) has been implicated in the resistance of cancer cells to cisplatin. Using a simple in vivo assay in bacterial culture, in the present study we demonstrate that ATP7B can confer resistance to cisplatin by sequestering the drug in its N-terminal metal-binding domain without active drug extrusion from the cell. Expression of a protein fragment containing four N-terminal MBRs (metal-binding repeats) of ATP7B (MBR1-4) protects cells from the toxic effects of cisplatin. One MBR1-4 molecule binds up to three cisplatin molecules at the copper-binding sites in the MBRs. The findings of the present study suggest that suppressing enzymatic activity of ATP7B may not be an effective way of combating cisplatin resistance. Rather, the efforts should be directed at preventing cisplatin binding to the protein.

While it is well-known that adsorbed proteins on implanted biomaterials modulate inflammatory responses, modulation of dendritic cells (DCs) via adhesion-dependent signaling has only been begun to be characterized. In this work, we demonstrate that adhesive substrates elicit differential DC maturation and adaptive immune responses. We find that adhesive substrates support similar levels of DC adhesion and expression of stimulatory and co-stimulatory molecules. Conversely, DC morphology and differential production of pro- and anti-inflammatory cytokines (IL-12p40 and IL-10, respectively) is adhesive substrate-dependent. For example, DCs cultured on collagen and vitronectin substrates generate higher levels of IL-12p40, whereas DCs cultured on albumin and serum-coated tissue culture-treated substrates produce the higher levels of IL-10 compared to other substrates. Additionally, our results suggest substrate-dependent trends in DC-mediated allogeneic CD4(+) T-cell proliferation and T-helper cell type responses. Specifically, we show that substrate-dependent modulation of DC IL-12p40 cytokine production correlates with CD4(+) T-cell proliferation and T(h)1 type response in terms of IFN-gamma producing T-helper cells. Furthermore, our results suggest substrate-dependent trends in DC-mediated stimulation of IL-4 producing T-cells, but this T(h)2 type response is not dependent on DC production of IL-10 cytokine. This work has impact in the rational design of biomaterials for diverse applications such as tissue-engineered constructs, synthetic particle-based vaccines and the ex vivo culture of DCs for immunotherapies.

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.

Using cupric phenanthroline as a cross-linking agent, we have shown that melittin induced time-dependent aggregations of Na,K-ATPase in microsomal fractions and in preparations of purified Na,K-ATPase from duck salt glands. Incubation of melittin with these preparations also led to the progressive loss of Na,K-ATPase activity. At melittin/protein molar ratio of 5:1, we did not observe inhibition of Na,K-ATPase in the microsomal fraction but the process of enzyme aggregation occurred. At higher melittin/protein molar ratios (10:1 and 30:1), the inhibition of the enzyme and its aggregation proceeded simultaneously but the rates of these processes and maximal values achieved were different. At a melittin/protein ratio of 30:1, Na,K-ATPase inhibition may be described as a biexponential curve with the values for pseudo-first order rate constants being 2.7 and 0.15 min(-1). However, the aggregation may be presented by a monoexponential curve with a pseudo-first order rate constant of 0.15 min(-1). In purified preparations of Na,K-ATPase, the maximal aggregation (about 90%) was achieved at a melittin/protein molar ratio of 2:1, and a further increase in the melittin/protein ratio increased the rate of aggregation but did not affect the value of maximal aggregation. The results show that melittin induced both aggregation and inhibition of Na,K-ATPase but these two processes proceeded independently.

Proteins interacting with alpha 1 beta 1-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.