摘要:

IntroductionParkinson’s disease (PD) is a degenerative neurological disorder caused by the loss of dopaminergic cells within the pars compacta region of the substantia nigra (SN).1Co-incident with the appearance of symptoms of the disease an elevation of iron has been observed within the SN.2–4Iron is suspected to be involved in the formation of reactive oxygen species within the SN, which is hypothesised to lead to the death of dopamine producing cells. It is unclear if the increased oxidative stress caused by Fe in PD is a cause or effect of the disorder.5A common mouse model for study of Parkinsonism is the 6-hydroxydopamine (6-OHDA) lesion method. The lesion is produced by directly injecting the neurotoxin into the region of the SN, where it is taken up by the neurons and kills the cells by the production of active superoxide radicals.6Fe is suspected to play a significant role in the mechanism of cell death and superoxide radical generation.2,3,7–9Development of methods to measure the regional concentration of trace metals in induced Parkinsonism models would be of benefit to probe the mechanism and treatment of PD. Solution nebulisation ICP-MS was employed by Tarohdaet al.10The SN was excised from 6-OHDA lesioned mouse models and analysed for Mn, Fe, Cu and Zn after closed-vessel microwave digestion. The concentration of Mn, Fe, Cu and Zn increased in the SN to a constant maximum at 7–10 days after injection of the neurotoxin. Solution ICP-MS has also been applied to monitoring iron levels in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated mice, another mouse model for Parkinsonism.11Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) may be utilised forin situanalysis of trace metals in biological tissue. Using a process we termelemental bio-imaging, isotope-specific maps of the spatial distribution of trace elements within thin tissue sections can be constructed. ICP-MS is an element analyser, designed to measure trace levels of the elements unlike other forms of “organic” MS that are used to identify and quantify molecular compounds. Laser ablation is a sample introduction system for ICP-MS that allow for the elemental composition of solid materials, including tissues, to be determined.The biggest limitation of LA-ICP-MS is a lack of reliable validated quantification strategies. Most studies have relied upon certified reference materials or preparation of matrix matched standards. Examples of the former include pig liver paste (LGC 7112) for single point calibration for quantification of trace elements in sheep liver.12Jacksonet al.used pressed pellets of TORT-2 (lobster hepatopancreas), DOLT-2 (dogfish liver) and DORM-2 (dogfish muscle) to build multi-point curves for quantification of Cu, Zn and Fe in rodent brains.13Matrix-matched standards have also been prepared by spiking brain tissue with known amounts of aqueous standards and ablating cut sections.14Elemental bio-imaging has also been applied to imaging of P, S and several transition metals in small sized tumours produced by F98 glioma cell implantation in rat brains.15,16Imaging of trace elements in both healthy and tumourous human brain tissue has also been reported.14,17–19This study presents a method to quantify the spatial and regional distribution of Mn, Fe, Cu and Zn in thin tissue sections taken from a 6-OHDA Parkinsonism mouse model, as well as untreated controls. The regional quantification of the substantia nigra obtained by the imaging method was also compared against that obtained by solution nebulisation ICP-MS.

ISSN:1756-5901    

DOI:10.1039/b816188g

出版商:RSC    

年代:2008

数据来源: RSC

摘要:

IntroductionDelivery of essential transition metals such as Cu, Zn, and Fe to the countless target sites inside a cell requires intracellular trafficking. In multicellular organisms there is, in addition, the need for cell-to-cell and long distance transport. Stable metal–chelator complexes are essential to suppress uncontrolled binding of metal ions to proteins and other biological molecules, for instance in plants during symplastic passage and during transport via xylem and phloem.1,2Candidate low-molecular weight metal chelators in plants are nicotianamine (NA), a non-proteinogenic amino acid synthesized through condensation of three molecules of S-adenosylmethionine by nicotianamine synthases (NAS),3glutathione (GSH) and phytochelatins (PCs), glutathione-derived metal-binding peptides,4and amino acids such as histidine.5Organic acids have also repeatedly been discussed as binding partners for transition metals.6However, metal–citrate or metal–malate complexes display low stability constants and are therefore likely to act as binding partners only in compartments such as vacuoles and the xylem which contain fewer competing molecules.7,8NA binds several transition metals with very high affinity. Logarithms of the stability constants are 18.8 for Cu(ii), 16.1 for Ni(ii), 14.8 for Co(ii), 14.7 for Zn(ii), 12.1 for Fe(ii), and 8.8 for Mn(ii).9Because the stability of complexes with most essential transition metals is much higher at pH values above 6.5,10NA is considered a symplastic chelator.11The exceptions are Cu(ii)–NA complexes which are also stable in an acidic environment down to pH 3. Numerous observations implicate NA in Fe and Cu homeostasis in plants.8More indirect evidence suggests a role in Zn and Mn homeostasis as well. NA deficiency causes intercostal chlorosis especially in young leaves of the tomato mutantchloronerva,3,12indicating defective Fe transport and possibly Zn transport to developing tissues. While long-distance transport of Fe is not affected inchloronerva, Cu content of shoots is strongly reduced,13consistent with complexation of Cu(ii) with NA in the acidic xylem sap as also suggested by a study of copper complexes in the xylem sap ofBrassica carinata.14In transgenic tobacco plants, made NA deficient through the overexpression of an NA aminotransferase, Fe, Zn, Mn, and Cu contents of leaves are reduced. AnA. thaliana NASquadruple mutant develops severe symptoms of Fe deficiency15and shows reduced levels of Zn in aboveground tissues. NAS overexpression in tobacco results in 2.5 fold higher Zn levels and 1.9 fold higher Fe levels in young leaves.16Other studies with NAS overexpressing tobacco andA. thalianaplants reported elevated Ni2+tolerance as the major phenotype.17,18Seeds of transgenic tobacco grown in a serpentine soil again contained more Fe, Zn, Cu, and Mn.19NA has also been implicated in metal hyperaccumulation in plants. Certain plant species are capable of accumulating metals such as Zn in leaves up to a level of 30 000 ppm, which is >100 fold more than in the tissues of non-hyperaccumulating plants.20The two most widely studied hyperaccumulation model systems areThlaspi caerulescensandArabidopsis halleri. In extracts of the former, Ni(ii)–NA complexes were detected by size exclusion chromatography coupled to ICP-MS.21Comparative transcriptome studies withA. thalianaandA. hallerifound that totalNASmessage and protein levels are strongly elevated inA. halleriroots and shoots relative toA. thaliana.22,23Similar observations were made forT. caerulescens.24Thus, elevatedNASexpression is part of the “hyperaccumulation syndrome”,i.e.the constitutive strong expression of metal homeostasis genes.25The expression difference inA. halleriroots resulted in constitutively higher NA levels. Formation of NA inS. pombecells rescued Zn2+hypersensitivity of a mutant strain unable to store excess Zn in the endoplasmic reticulum.23Based on these observations and the above-mentioned phenotypes of transgenic plants with reduced and elevated levels of NA16it was proposed that (i) NA plays a role in Zn homeostasis as an intracellular Zn chelator and that (ii) higher NA levels contribute to Zn hypertolerance and hyperaccumulation inA. halleriandT. caerulescens.Until recently, NA had been considered a plant-specific metabolite. However, filamentous fungi such asNeurospora crassa,Magnaporthe griseaandPodospora anserinacarry putativeNASgenes as apparent from their genome sequences. ForN. crassait was indeed demonstrated that it synthesizes NA and expresses a functionalNASgene which is massively up-regulated under conditions of Zn deficiency.26Most of the available evidence for the functions of NA in metal homeostasis is indirect. Detection and quantification of metal–ligand complexesin vivo,i.e.metal ion speciation analysis, is still extremely difficult because tissue disruption immediately changes the chemical environment and thereby the availability of binding partners.8Non invasive techniques, such as X-ray absorption spectroscopy (XAS), have been applied to trace element accumulations in plants.27Here the weak association of metal ions with organic acids in vacuoles, and not binding to strong ligands, was found to be the main storage form of metals and the metalloid arsenic.28–34Recently, the focus of the studies has been expanded to cell compartments.35Here we analyzeS. pombecells, in which we modulated metal ligand production to learn more about thein vivoformation of metal–NA complexes. A plantNASgene was expressed in various mutant strains. The question of Zn(ii)–NA complex formation was addressed by subjecting suchS. pombecells to analysis by X-ray absorption spectroscopy (EXAFS and XANES) in a frozen-hydrated state, thus avoiding artefactual Zn(ii)–NA complex formation during sample preparation. Further, by analyzing previously measured32Zn–EXAFS spectra ofT. caerulescenswe tested whether, in hyperaccumulators, NA binds a major proportion of the accumulated Zn,i.e.is involved in Zn storage, or rather acts as a transport ligand, in which case only a small proportion of the total Zn would be bound to NA.

ISSN:1756-5901    

DOI:10.1039/b913299f

出版商:RSC    

年代:2009

数据来源: RSC

摘要:

IntroductionArsenic, a hazardous metalloid, is the inorganic component of a chemical warfare agent named Blue Cross, which contains diphenylchloroarsine (DA, Clark I) and diphenylcyanoarsine (DC, Clark II). These agents were mainly used during World War I as vomiting agents; the main purpose was to be “mask breakers” by penetrating the canister; thereby forcing the opposing troops to remove their masks and be further exposed to toxic agents. A concern with these compounds is the possibility of prolonged systemic effects, such as headache, mental depression, chills, nausea, abdominal cramps, vomiting and diarrhea, all lasting for several hours after exposure. The agents are dispersed as aerosols and they produce their effects by inhalation or by direct action on the eyes. When released indoors, they can cause serious illness or death.1,2Chemical warfare agent degradation products (CWDPs) include diphenylarsinic acid (DPAA), phenylarsonic acid (PAA) and phenylarsine oxide (PAO). In the degradation pathway, byproducts such as triphenylarsine (TPA) and triphenylarsine oxide (TPAO) are found and their structures are shown inFig. 1. Previous papers have mentioned that groundwater and soil have been contaminated from some of these organoarsenical compounds, leading to environmental problems.3,4Oyamaet al.reported as recently as 2007 the presence of degradation products DPAA, PAA and PAO in groundwater and soil in Kamisu City, Japan.5With known population exposure, it is important to understand the biological uptake mechanism for these degradation products in mammalian organisms. Examples of other studied arsenic species include sodium arsenate, Na2HAsO4[As(v)], sodium arsenite, NaAsO2[As(iii)], dimethylarsinic acid (DMA) and methylarsonic acid (MMA) as shown inFig. 1as well as parent agents Clark I and Clark II.Structures of primary warfare agents, their degradation products and other arsenic species used in the cytotoxicity study.The aim of this work was to do an initial study investigating CWDPs cytotoxicity on mammalian cells by comparing the effects of different CWDP concentrations over different time periods. To generate metallomics information an As speciation study was performed utilizing liquid chromatography (LC) coupled to both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization mass spectrometry (ESI-MS) to identify the molecular level changes the CWDPs might undergo.The cytotoxicity evaluation was conducted on an African Green Monkey CV-1 cell line based on quantification of lactate dehydrogenase activity as released from damaged/dying cells. Kidney cells were chosen as target cells because kidneys take up and excrete a variety of substances produced by the cell metabolism. A comparison of different concentration levels over different time ranges was completed, following the toxic compounds addition to the cells. The cells treated with the various arsenic species were then tested for cellular arsenic uptake by ICPMS detection of the monoisotopic75As.The interest in arsenic speciation analyses continues to grow with the increasing need to assess its biological effects. Total arsenic analyses, though necessary, are insufficient to fully address the complexities and questions posed by biological systems. While As totals are useful, they provide no information on the various arsenic forms, which have widely varying toxicities. The inorganic species, which are commonly found in the environment, are known to be the most toxic species, while some of the organic arsenic forms are less toxic and even innocuous.6Hence, speciation analysis is required and further identification and verification by molecular mass spectrometry as necessary.

ISSN:1756-5901    

DOI:10.1039/b816980b

出版商:RSC    

年代:2008

数据来源: RSC

摘要:

IntroductionAccording to the World Health Organization’s prediction in 2006, the number of type 2 diabetic patients in the world could increase to 360 million by 2030.1The goal of diabetes treatment is to control the blood glucose levels, body weight, blood pressure, and cholesterol and triglyceride levels, and prevent the development of complications.2Combinations of alimentotherapy, ergotherapy, oral antidiabetic medicines, and/or insulin have been used to treat diabetes mellitus. Among the oral antidiabetic medicines, α-glucosidase inhibitors inhibit α-glucosidase, which metabolizes disaccharides into monosaccharides in the small intestine. The inhibition of α-glucosidase delays the digestion and absorption of carbohydrates, resulting in suppression of postprandial hyperglycemia and excessive insulin secretion. Thus, α-glucosidase inhibitors lower the insulin requirement resulting from less absorption of glucose, and lead to less strain on pancreatic β-cells in insulin production. Hyperinsulinemia is considered as a potential risk factor for arteriosclerosis. Reduced requirement for insulin secretion can reduce the risk of arteriosclerosis.3α-Glucosidase inhibitors including acarbose,4–7voglibose,8,9and miglitol10–13have been available for clinical use since 2005 (Fig. 1). Such α-glucosidase inhibitors have been approved not only as medicines but also as a food for specified health uses (FOSHU), that is available without prescription.14–16Structures of clinically used α-glucosidase inhibitors.Several metal ions and their complexes exhibit antidiabetic effects.17–22A chromium-containing material extracted from pig spleen improved glucose tolerancein vivo.17Manganese plays an important role in glucose metabolism.18Zinc exerts an insulin-like effect in rat adipocytes.19In addition, some metal ions, such as tungsten,23vanadium,24and selenium25lower high blood glucose levels in the diabetic state. It appears attractive to many researchers to study the relationship between diabetes mellitus and metal ions. The action mechanism of these metal ions, however, remains unclear. We have examined the mechanism of some transition metal ions and their complexes since 2006 and demonstrated that they activate the insulin signaling pathway followed by glucose transporter 4 (GLUT4) translocation to the plasma membrane and the enhancement of glucose utilization.26–29Furthermore, in 2004, it was reported that insulin/C-peptide were closely associated with metal ions.30In this study, we have focused on the inhibitory activity of these metals on α-glucosidase to analyze alternative action mechanisms of these metal ions. We have evaluated the α-glucosidase inhibitory activity of several divalent first-row transition metal ions, whose antidiabetic effects have been reported, such as vanadium, manganese, iron, cobalt, nickel, and copper, and a non-transition metal ion, zinc,24,31,32together with divalent alkaline earth metal ions such as magnesium, calcium, strontium, and barium.33In addition, we have compared their inhibitory activities with α-glucosidase inhibitor, acarbose, used for clinical purposes in bothin vitroandin vivoexperimental systems, and then evaluated the mode of α-glucosidase inhibition.

ISSN:1756-5901    

DOI:10.1039/b906709d

出版商:RSC    

年代:2009

数据来源: RSC

摘要:

IntroductionThe 90% survival rate of patients with testicular cancer highlights the effectiveness of the widely used chemotherapy drug cisplatin,cis-diamminedichloro-platinum(ii).1Although cisplatin and later-generation Pt drugs carboplatin and oxaliplatin (seeChart 1) are also used against other cancers, several drawbacks limit the survival rate observed in testicular cancer. In many cases, treatment is diminished by side effects of nephrotoxicity and neurotoxicity, as well as inherent and acquired resistance of many cancer types to this class of drug. Understanding the molecular basis for resistance is critical to discovering new treatment regimens in order to expand the effectiveness of these drugs.Clinically important Pt drugs.Cisplatin is administered as a formulation that includes 154 mM NaCl that suppresses hydrolysis of the chloride ligands from platinum. The high chloride ion concentration in the blood (∼105 mM) presumably allowscis-[Pt(NH3)2Cl2] to remain neutral until in enters the cytosol. Here it encounters a 4 mM chloride ion concentration which facilitates hydrolysis of the chloro ligands to form aquated [Pt(NH3)2(OH2)Cl]+, [Pt(NH3)2(OH)Cl], [Pt(NH3)2(OH2)2]2+, and [Pt(NH3)2(OH)2] species. The aquo and hydroxo forms are more reactive towards nucleophiles than the original chloro species, and upon entering the nucleus they bind to N7 positions on adjacent guanine bases of DNA. These intrastrand DNA cross-links are the primary biological targets that lead to cell death.1Any side reaction that diverts thecis-Pt(NH3)22+core from forming these toxic lesions on DNA is a potential pathway for drug inactivation, increased side effects, or resistance. Such pathways include inactivation of the Pt compounds by sulfur-containing molecules like glutathione, increased repair of the cell-killing Pt–DNA adducts, and decreased drug accumulation.2Of these mechanisms, the decreased accumulation of Pt drugs is the most common defect found in resistant cells and therefore demands critical attention in order to maximize the effectiveness of this well proven class of drugs across a wider range of cancer types.Observations that cisplatin uptake is not saturable, does not have a pH optimum, and is not inhibited by structural analogs lead to the long-standing hypothesis that cisplatin passively diffuses across cell membranes.1While passive diffusion remains a viable mechanism for Pt drug entry, current estimates suggest that only half of Pt drug uptake isviapassive diffusion, while the remainder enters by other transport mechanisms that are only beginning to be discovered and appreciated.3,4One of these is the cell-surface copper transport protein Ctr1.5Increasing and compelling evidence is revealing that cellular processes associated with Cu homeostasis are modulating Pt accumulation, particularly in resistant cells.6A connection between Cu and Pt trafficking was suggested by observations of bidirectional cross-resistance of Pt drugs and Cu in a variety of cell lines.5,7Cells selected for resistance to high levels of Cu were found to be resistant to Pt drugs; likewise, highly Pt-resistant cell lines were found to be moderately resistant to Cu.8The cytotoxic properties of these two metals are quite different, implying that their similar phenotype derives from impairment in shared uptake or efflux mechanisms. Work in yeast and mammalian cells identified the shared uptake mechanism as the transmembrane copper transporter Ctr1,9and both ATP7A and ATP7B, two structurally similarP-type ATPases that mediate Cu efflux from mammalian cells, have been implicated in removing Pt drugs from cells.5,7,10,11Yeast cells in which the CTR1 gene is deleted show increased resistance to cisplatin that correlates with reduced uptake of cisplatin and reduced formation of the critical Pt–DNA adducts compared with control cells.9,12Cisplatin has been shown to induce internalization and degradation of hCtr1 as well as impede the cellular uptake of both carboplatin and oxaliplatin.13,14These initial reports clearly establish a biological link between Ctr1 and Pt drugs; however, how cisplatin interacts with Ctr1 and how the two molecules modulate each other’s trafficking and function is unclear. Recent work from the Howell lab using murine embryonic fibroblasts in which both CTR1 alleles were deleted indicates that the copper transporter controls cellular accumulation of cisplatin, carboplatin, and oxaliplatin. Subsequently, Howellet al.observed that oxaliplatin at higher concentrations may use a different cellular entry mechanism.15In addition to these conventional mononuclear Pt drugs, an experimental polynuclear Pt compound BBR3464 has also been demonstrated to utilize hCtr1 to gain cellular access.16Studies of a variety of human cell lines in which hCtr1 is over-expressed reveal that simply increasing the level of hCtr1 does not always correlate with an increase in cell death by Pt drugs. For example, human ovarian carcinoma cells engineered to over-express hCtr1 accumulate more Pt than their counterparts, but this over-accumulation does not lead to increased Pt–DNA adducts or cisplatin sensitivity.7This implies that not all Pt drugs brought inviaa Ctr1-mediated pathway reach their DNA target. Not all cisplatin-resistant cell lines show reduction in endogenous Ctr1 expression levels compared with cisplatin-sensitive counterparts.17Similarly, at least one Pt-resistant cell line, a cervix squamous cell carcinoma, does not accumulate increased levels of cisplatin when it is engineered to over-express hCtr1.18However, because it is not clear that over-expressed Ctr1 functions normally in human cells, it is not at all obvious that its forced over-expression should be expected to lead to an increase in Pt accumulation.15More conflicting results describe the trafficking of Ctr1 in the presence of Pt drugs. In human ovarian carcinoma cells that endogenously or exogenously express hCtr1, cisplatin causes rapid disappearance of the transporter from the plasma membrane, suggesting that cisplatin down-regulates its own transporter in these cells.19On the other hand, Ctr1 expressed in human embryonic kidney cells is stabilized at the plasma membrane as a multimeric complex, and is not endocytosed or degraded upon Pt treatment.20These seemingly conflicting results expose the need to more fully understand the factors governing Pt drug access to cells in general and Pt–Ctr1 interactions in particular.Human hCtr1 contains three transmembrane regions that situate the amino terminus on the extracellular side with the carboxyl terminus facing the cytosol.21–24The protein likely exists as a homotrimer in its active form.21,25–27The unique feature of the amino-terminal region of Ctr1, which is the first gate of entry for cellular copper, is the presence of methionine-rich domains arranged as MXXM or MXM motifs containing 3–5 methionine residues per “Mets” motif. Yeast yCtr1 has 8 Mets motifs, with a total of 30 methionine residues in the ∼140-residue extracellular domain, whereas human hCtr1 has two Mets motifs in a 65-residue extracellular region.The extracellular N-terminal region of Ctr1 is critical for Ctr1-mediated Pt drug entry into cells. Pt drug accumulation studies with hCtr1 deletion mutants show that full-length and C-terminally truncated protein both transport Pt drugs, whereas an N-terminal deletion mutant fails to do so.17,28The N-terminal Mets motifs were also found to be responsible for stabilizing multimeric hCtr1 at the plasma membrane in HEK cells expressing hCtr1.20Mutagenesis of the eight methionines that make up the two Mets motifs of hCtr1 to alanines results in protein that appropriately traffics to the plasma membrane but does not trimerize upon cisplatin exposure, suggesting that the stable trimers observed in the parent protein result from cisplatin cross linking adjacent proteinsviamethionine binding. Further evidence for this model is provided by the fact that trimer formation is reversed upon exposure to Pt chelating compounds.20A study that used fluorescent fusion proteins to monitor molecular dynamics of yCtr1 found that Cu(i) induced FRET but that cisplatin, which accumulated in a Ctr1-dependent manner, did not.28This result highlights the fact that differences in the chemistry of copper and platinum translate into differences in metal-induced protein conformational changes and mechanisms of metal transport by Ctr1.Several lines of evidence point to Cu(i) as the active redox state for Ctr1,21,26,29–31and we have previously shown that isolated Mets domains are capable of binding Cu(i) selectively over Cu(ii).32Additionally, yCtr1 is unable to transport Zn(ii) or other divalent metal ions but readily takes in Ag(i) as a surrogate for Cu(i).27Given the fact that Ctr1 does not transport divalent metal ions and that it seems to be ideally suited for transporting soft, kinetically labile, monovalent cations, it would actually be quite surprising that this protein transports intact Pt(ii) coordination compounds. Apart from the fact that both Cu(i) and Pt(ii) prefer soft, sulfur-rich coordination environments, their coordination chemistry otherwise is quite different. Whereas Cu(i) can adopt linear, trigonal, or tetrahedral coordination geometries, Pt(ii) is rigorously square planar. In addition, the ligands on Pt(ii) are less likely to be kinetically labile, although inner-sphere ligand exchange reactions can occur, with their likelihood depending on thetranseffect of the ligands.The affinity of Pt(ii) for sulfur-containing ligands has provoked a long-standing question of how Pt drugs reach their DNA targets in spite of significant competition for binding to S-containing amino acids and proteins.33,34Because neuro- and nephrotoxicity associated with Pt drug treatment has been attributed to S-containing proteins, and because intracellular S-containing compounds like glutathione may act as intracellular drug reservoirs,33numerous studies have been done to describe the interactions of simple amino acids like cysteine, methionine, and glutathione with various Pt(ii) compounds.35–42These studies have revealed that the thioether of methionine can displace chloro or aqua ligands of cisplatin. Thioether sulfur has a strongtransinfluence, therefore methionine coordination to square-planarcis-diaminePt(ii) core can result in loss of the amine ligandtransto the incoming thioether. Many model studies have used stable tridentate chelates of diethylenetriamine (dien) in order to suppress ligand loss and focus exclusively on the Pt–S bonding interaction.43–49However, the potential replacement of the pendant ligands on Pt drugs will have important repercussions on the mechanism of action of Ctr1-transported Pt, since thecis-diamminePt(ii) core is the active metal complex that binds to DNA to affect its cytotoxic action. Therefore we focus here exclusively on the interactions of hCtr1 peptides with the clinically important drugs themselves. Methionine–platinum adducts are not unique to human Ctr1 species as work by Arnesanoet al. have shown that in the presence of a methionine-rich sequence of yCtr1, cisplatin is stripped of its ligands and binds in a 1 : 1 ratio.50,51Certain Pt(ii) complexes selectively hydrolyze peptide amide bonds by first anchoring onto Met or His side chains.52–54Cisplatin is included in this family of inorganic hydrolases, and has been shown to cleave the second amide bond upstream from an anchoring Met in relatively simple acetylated di- and tripeptides.55–57These model studies strongly encourage the examination of peptides containing multiple Met residues such as those found in hCtr1 to determine if hydrolysis is a possible mechanism of action.The differences in the coordination chemistry of Cu(i) and Pt(ii) raise two fundamental questions related to Ctr1-mediated Pt drug uptake: (1) What happens to Pt drugs upon interacting with the extracellular domains of Ctr1? Our hypothesis is that methionine residues in the Mets domains induce ligand exchange reactions that change the molecular speciation of the original drug, which has important ramifications for its overall efficacy. (2) How does the interaction of the protein with Pt drugs change the integrity of the protein itself? Given the proclivity of Pt compounds to hydrolyze amide bonds, it is possible that Pt drugs may degrade hCtr1. To gain insight into these questions, we examined the reaction products of model Mets peptides derived from human hCtr1 with Pt drugs by combined liquid-chromatography–mass spectrometry (LC–MS).

ISSN:1756-5901    

DOI:10.1039/b916899k

出版商:RSC    

年代:2009

数据来源: RSC

摘要:

IntroductionSelenium (Se) is an essential micronutrient in animals because it promotes the activities of such selenoenzymes as glutathione peroxidases, iodothyronine 5′-deiodinase, and thioredoxin reductase, by existing in the form of selenocysteine (SeCys) in their active centers.1,2In contrast, Se is not an essential element in plants; rather, it is a serviceable element for growth, similar to sodium and silicon. In spite of its non-essentiality, Se metabolism in plants has been attracting much interest for two reasons. One, is its used in phytoremediation for the removal of contaminating Se. Although Se is a useful material in the semiconductor and electronic industries, it is also an environmental contaminant.3Phytoremediation is a low-cost and environmentally friendly technique to remove Se contaminants. Indeed, the Indian mustard,Brassica juncea, has been extensively studied for Se phytoremediation4–6because of its high Se accumulating ability, fast growth, and high biomass. In this plant, absorbed inorganic Se, such as selenite (Se (iv)) and selenate (Se (vi)), is transformed into less toxic forms,i.e., selenoamino acids, such asSe-methylselenocysteine (MeSeCys) and γ-glutamylmethylselenocysteine (GluMeSeCys). The other reason is its use as a nutritional aid in the form of Se supplements or medicines for cancer prevention and other diseases. As inorganic Se salts exhibit substantial toxicities to animals, less toxic Se compounds from plants are useful for this purpose. Indeed, it has been reported that some selenoamino acid derivatives decrease the incidence of prostate, skin, and mammary cancers7–9and exert cytotoxic effects on cultured tumor cells.10–12However, Se metabolism in plants is not fully revealed, and novel selenopeptides and selenometabolites have been reported with the development of analytical techniques based on mass spectrometry.13,14Selenomethionine (SeMet) is one of the key metabolites in selenized yeasts and plants.15–18In selenized yeast, SeMet biosynthesized from inorganic Se is incorporated into proteins and metabolized to other biomolecules such asSe-adenosylselenomethionine (AdoSeMet) derivatives.19,20As already suggested, the replacement of methionine (Met) with SeMet did not significantly alter protein structure in bacteria and fungi.21,22In addition, we have also reported that SeMet, which could not be discriminated from Met, was incorporated into proteins in anin vitrotranslation system using wheat germ extract (WGE),23also, the fluorescence of green fluorescent protein (GFP) and the activity of JNK stimulatory phosphatase-1 (JSP-1) were not affected by the substitution of Met with SeMet byin vitrotranslation.23Thus, the biosynthesis of SeMet-containing proteins is responsible for the detoxification of Se in plants in addition to the biosynthesis of MeSeCys and GluMeSeCys.24On the other hand, although Met is used not only for protein synthesis but also metabolism into coenzymes, such as methylmethionine (MeMet) andS-adenosylmethionine (AdoMet, SAM), as shown in yeast, the metabolism of SeMet into such coenzymes in higher eukaryotes,i.e., plants and animals, has yet to be revealed.Although the inductively coupled plasma mass spectrometer (ICP-MS) is a superior instrument for elemental speciation owing to its high sensitivity and specificity when used in combination with separation instruments such as a high-performance liquid chromatograph (HPLC), the identification of Se compounds by HPLC-ICP-MS is accomplished by matching the retention times of samples with those of authentic standards.25,26Thus, identification by HPLC-ICP-MS is limited to the situation where authentic Se standards are available. As a complementary method to HPLC-ICP-MS, HPLC coupled with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS/MS) is used to identify unknown selenometabolites. Indeed, novel Se-containing metabolites, such as selenosugars, selenoamino acid derivatives, and AdoSeMet derivatives, have been successfully identified with this method.20,27–31In this study, we intend to reveal the biological transformation of SeMet into SeMet derivatives in a model system of higher eukaryotes,i.e., WGE, by identifying Se-containing molecules with HPLC-ICP-MS and HPLC-ESI-MS/MS. As mentioned above, WGE is a good model system to reveal the metabolism of SeMet. In addition, thein vitrometabolism of SeMet was also evaluated in rabbit reticulocyte lysate (RRL), a mammalianin vitrotranslation system. Moreover, we also intend to depict the entire metabolic pathway of SeMet in higher eukaryotes by assigning Se metabolites on the basis of selenometabolomics.

ISSN:1756-5901    

DOI:10.1039/b813118j

出版商:RSC    

年代:2008

数据来源: RSC

摘要:

IntroductionIn the 21st century, the prevalence of metabolic diseases, including diabetes mellitus, is markedly increasing worldwide.1Diabetes mellitus is classified into two major types: type 1, insulin-dependent diabetes; and type 2, noninsulin-dependent diabetes.2Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic β cells leading to a lack of intrinsic insulin secretion, and sufferers consequently require daily insulin injections for survival. Type 2 diabetes, accompanied by obesity, impaired glucose metabolism, and insulin resistance, requires treatment with hypoglycemic or anti-diabetogenic synthetic compounds together with diet control and exercise.3,4We have developed a novel chemical compound that can enhance the lowering of blood glucose and we have examined the action mechanisms of the compound in diabetic animals.Vanadium, a trace element in animals and humans, has a wide variety of biological and physiological functions.5Vanadyl (VO2+, +4 oxidation state) and vanadate (H2VO4−) complexes have insulin-mimetic, anti-tumorigenic and anti-osteogenic activities.6–9In particular, vanadyl complexes with several coordinating environments around the vanadyl ion are candidate agents to treat the hyperglycemic state in animals and humans.6,9Vanadyl is known to be less toxic than vanadate.5,6Vanadyl complexes, bis(picolinate)oxidovanadium(iv) [VO(pa)2] and bis(maltolato)oxidovanadium(iv) [VO(ma)2] given by intra-peritoneal injection, show the ability to reduce high blood glucose levels in streptozotocin (STZ)-induced diabetic animals.6,9–11Vanadium enhances tyrosine phosphorylation of the insulin receptor β-subunit (IRβ) and the insulin receptor substrate (IRS) by inhibiting protein tyrosine phosphatase 1B (PTP1B), which in turn activates the signaling pathways of phosphatidylinositol 3-kinase (PI3K)-Akt (also known as protein kinase B).12Both Akt and GSK3β are important transmitters of the insulin signaling that regulates glucose metabolism.13The activation of these signals stimulates glucose uptake, glycogen synthesis and lipogenesis, but inhibits lipolysis and gluconeogenesis.14Glucose transporter 4 (GLUT4) is mainly expressed in insulin-responsive tissues, such as skeletal muscles and adipose tissues.13The activation of the translocation of GLUT4 to the cell surface is important for glucose utilization. In the diabetic state, both protein and mRNA levels of GLUT4 are down-regulated.15,16Oral administration of VO(ma)2increased GLUT4 expression level in the skeletal muscles of STZ-rats.16Insulin controls gene transcription by regulating the activation or suppression of transcription factors.17Among them, the forkhead box transcription factor class O (FoxO) family and the sterol-response-element-binding protein (SREBP) family regulate transcriptional activities in the presence of insulin.17These transcription factors regulate genes involved in glucose and lipid metabolism. Genes associated with β-oxidation, lipid saturase, and oxidative stress increased expression levels in the skeletal muscles or kidneys of diabetic mice and rats,15,18–22while GLUT4, hexokinase II and the genes of the mitochondrial electron transport chain in the insulin action pathways were down-regulated.15Efficacy of the vanadyl complexes on gene expression, however, remains unclear, especially when they are given orally.Bis(allixinato)oxidovanadium(iv) [VO(alx)2] (Fig. 1A) is one of the most effective vanadyl complexes for lowering hyperglycemia by intra-peritoneal administration in both the STZ-induced diabetic and the obese type 2 diabetic KKAymouse models.23,24VO(alx)2enhanced the level of phospho-protein in the insulin signaling and induced the GLUT4 to the cell surfacein vitro.25The oral administration of this complex not only improved hyperglycemia, but also normalized hypertension and leptin level in the KKAymice. Thus, it indicates that VO(alx)2pharmacologically improves both diabetes and metabolic diseases. In the present study, we have aimed to examine whether the oral administration of VO(alx)2lowers high blood glucose levels in the STZ-diabetic mice and stimulates the insulin signaling pathwayin vivoleading to improvement of diabetes. Furthermore, we have examined gene expression profiles to compare the effects of insulin and VO(alx)2treatments on the skeletal muscles of the diabetic mice.Improvement of hyperglycemia in STZ-diabetic mice following oral administration of VO(alx)2. (a) Structure of VO(alx)2. (b) Changes in blood glucose level in STZ-diabetic mice treated with insulin (1 U kg−1body weight) by injection or VO(alx)2(7 mg (137 μmol) V kg−1body weight) by oral administration for 9 days (n= 4 to 7 mice/group). The symbols indicate the following: closed squares, non-diabetic control mice; closed triangles, STZ-diabetic mice; closed circles, insulin-treated STZ-diabetic mice; open circles, VO(alx)2-treated STZ-diabetic mice. Data are expressed as means ± SD. Significance: *P< 0.01versusbefore treatment.

ISSN:1756-5901    

DOI:10.1039/b815384c

出版商:RSC    

年代:2008

数据来源: RSC

摘要:

IntroductionMercury (Hg) pollution is a ubiquitous problem resulting both from natural events and anthropogenic activities. The amount of Hg mobilized and released into the biosphere has gradually increased since the beginning of the industrial age. It has caused deep concern because of its toxicity, mobility, bioaccumulation, methylation process and transport in the biosphere.1When absorbed by human bodies, it causes neurological toxicity, kidney damage and even death due to its strong and specific interaction with sulfhydryls in proteins.2,3Hg-contaminated soil is believed to contribute to human health risks and major environmental problems. Many studies have shown that plant roots accumulate Hg when exposed to Hg-contaminated soils.4–6As a typical soft Lewis acid, Hg2+complexes strongly with reduced sulfur-containing ligands resulting in the predominant Hg chemical form in aquatic and soil environments.7,8Humic acids (HA), the predominant fraction of humic substances and well known Hg ligands with cysteine (Cys) in their peptide fragments, tend to increase Hg solubility and mobility, and alter its availability to plants.9,10When exposed to heavy metal ions including Hg2+, higher plants andSchizosaccharomyces pomberespond by synthesizing Cys sulfhydryl residue-rich peptides and phytochelatins (PCs) in the cytoplasm to defend against their phytotoxicity.11,12PCs have the general structure of γ-(Glu–Cys)n–Gly (n= 2–11), and are synthesized from glutathione (GSH) through a constitutively present PC synthase. Subsequently, heavy metal ions such as Hg2+are complexed and sequestered by the induced PCsviathiolate coordination due to their high affinity with SH groups.13Previousin vitrostudies demonstrate that Hg2+is facile to transfer from shorter- to longer-chain PCs. The strength of Hg2+binding to GSH and PCs follows the order γ-Glu–Cys–Gly < γ-(Glu–Cys)2–Gly < γ-(Glu–Cys)3–Gly < γ-(Glu–Cys)4–Gly,12and GSH and PCs play important roles in resistance inHydrilla verticillata(l.f.) Royle andVallisneria spiralisL. under Hg2+stress.14On the other hand, it has been shown that a mutant having a defect in PC synthesis shows significantly enhanced sensitivity to Hg2+.15In addition, overexpression ofEscherichia coliγ-GlyCys synthetase and GSH synthetase inArabidopsis thalianaplants provides significant increases in tolerance and accumulation of Hg2+.16However, definite evidence ofin vivoHg–PC complexes and their corresponding PC precursors in support of the plant’s defense and accumulation mechanisms is still scarce, and this makes it difficult to understand the nature of the Hg–PC complexes present in plant tissues. Among the few studies found in the literature,12,14,17only one reports the detection ofin vivoHg–PC complexes inBrassica napus, but only PC2and its Hg complexes are observed in the case of adding chelating agents.17Since HA is the active fraction of soil organic substances and Cys is the most active component towards Hg in HA,18in this study we investigated the behavior of Cys and HA on Hg accumulation inBrassica chinensisL., and the subsequent synthesis of PCs and the formation of their corresponding Hg complexes for the first time. The hyphenation of RPLC with ESI-MS/MS was used to characterize not onlyin vitrosynthesized Hg–PC complexes so as to predict the possible Hg–PC complexes and their binding stoichiometry, but alsoin vivoHg–PC complexes present in the Hg-treated plant tissues to provide clear and definite evidence of detoxifying and/or deactivating Hg species by PCs in plant tissues.

ISSN:1756-5901    

DOI:10.1039/b815477e

出版商:RSC    

年代:2008

数据来源: RSC

摘要:

Metallomicspublishes original research and topical reviews, which provide insight into the role of metals in the life sciences. This includes:• Chemical speciation, dynamics and kinetics of trace elements in biological systems• Elemental distributions and concentrations linked to the genome• Regulation of the uptake, accumulation and metabolism of metals and other trace elements in biological systems• Physiological and pathological mechanisms related to trace elements in human health and disease• Structural analysis and coregulation of elements within the metallome, including the survey and identification of metalloproteins/enzymes• Genetic and molecular genetic basis for regulation of metallomes and epigenetic factors relative to the organism• The interaction of metallodrugs, including chemotherapy agents, with biological organisms, including in clinical use• Metal exchange between biota and the environment• Bioimaging and biosensing of metals, including analysis of diagnostic and therapeutic radioactive metals• Certified reference materials for biological applicationsReadership will be cross-disciplinary and include researchers in academia and industry working in analytical science, biogeochemistry, bioinformatics, biological catalysis, biological environmental science, cell biology, clinical chemistry, environmental health, medicine, metallobiochemistry, microbiology, nutritional chemistry, pharmacology, plant biochemistry and physiology and toxicology.

ISSN:1756-5901    

DOI:10.1039/c001365j

出版商:RSC    

年代:2010

数据来源: RSC

ISSN:1756-5901    

DOI:10.1039/c001179g

出版商:RSC    

年代:2010

数据来源: RSC