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UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4's subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the protein's possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation. Show less

AbstractBACKGROUND:Gold (III) compounds have exhibited favorable antitumor properties both in vitro and in vivo. In a previous study, the authors reported that the novel gold (III) complex 1a (gold 1a) exhibited strong cytotoxicity in some tumor cell lines. In the current study, the effect of gold 1a was investigated on colon cancer cells.METHODS:The cytotoxicity of gold 1a was determined by using the 3‐(4,5‐dimethyl‐2‐thihazyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide method. Flow cytometry was used to detect apoptosis and cell cycle. The expression of protein was evaluated by Western blot assay. Tumor growth in vivo was evaluated in nude mice.RESULTS:Gold 1a exhibited marked cytotoxic effects in vitro to human colon cancer, and the concentration of drug required to inhibit cell growth by 50% compared with control (IC50) values ranged from 0.2 μM to 3.4 μM, which represented 8.7‐fold to 20.8‐fold greater potency than that of cisplatin. Gold 1a significantly induced apoptosis and cell cycle arrest and cleaved caspase 3, caspase 7, and poly(ADP‐ribose) polymerase; released cytochrome C, and up‐regulated p53, p21, p27, and Bax. In vivo, intraperitoneal injection of gold 1a at doses of 1.5 mg/kg and 3.0 mg/kg significantly inhibited tumor cell proliferation, induced apoptosis, and suppressed colon cancer tumor growth. An acute toxicology study indicated that gold 1a at effective antitumor concentrations did not cause any toxic side effects in mice.CONCLUSIONS:The current results suggested that gold 1a may be a new potential therapeutic drug for colon cancer. Cancer 2009. © 2009 American Cancer Society. Show less

A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser-scanning confocal microscopy. The microscopy images indicated that complex 3a was localized in the perinuclear region upon interiorization. Temperature-dependence experiments suggested that the internalization of the complex was an energy-requiring process such as endocytosis. This has been confirmed by cellular-uptake experiments involving the luminescent conjugates Ir-BSA and Ir-TF (TF = holo-transferrin), which were prepared by conjugation of the proteins with the complex [Ir(pq)(2)(phen-NCS)](PF(6)) (phen-NCS = 5-isothiocyanato-1,10-phenanthroline). Show less

The synthesis and characterization of three novel iridium(III) complexes and one rhodium(III) complex with 1-nitroso-2-naphthol (3) chelating as a 1,2-naphthoquinone-1-oximato ligand are described. The reaction of mu(2)-halogenido-bridged dimers [(eta(5)-C(5)Me(5))IrX(2)](2) [X is Cl (1a), Br (1b), I (1c)] and [(eta(5)-C(5)Me(5))RhCl(2)](2) (2a) with 3 in CH(2)Cl(2) yields the mononuclear complexes (eta(5)-C(5)Me(5))IrX(eta(2)-C(10)H(6)N(2)O) (4a, 4b, 4c) and (eta(5)-C(5)Me(5))RhCl(eta(2)-C(10)H(6)N(2)O) (5a). All compounds were characterized by their (1)H and (13)C NMR, IR, and mass spectra, UV/vis spectra were recorded for 4a and 5a. The X-ray structure analyses revealed a pseudo-octahedral "piano-stool" configuration for the metals with bidentate coordination through oximato-N and naphthoquinone-O, forming a nearly planar five-membered metallacycle. The metal complexes 4a and 5a were evaluated in respect to their cytotoxicity and binding affinity toward double-stranded DNA. As determined in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, both exerted a much stronger cytotoxic effect toward HeLa and HL60 cancer cell lines than did cisplatin. The remarkable cytotoxicity of the compounds tested may be attributed to necrosis, rather than to apoptosis, as it is evidenced by the caspase-3/7 activation assay. No clear evidence was found for interaction with double-stranded DNA. The melting experiments showed no significant differences between thermodynamic parameters of intact DNA and DNA incubated with 3, 4a, or 5a, although these derivatives altered DNA recognition by the BamHI restriction enzyme. Therefore, the screened iridium and rhodium complexes 4a and 5a may still be interesting as potential anticancer drugs owing to their high cytotoxicity toward cancer cell lines, whereas they do not modify DNA in a way similar to that of cisplatin. Show less

The Rh(III) polypyridyl complexes of the type [RhCl(pp)([9]aneS(3))](2+) [(pp)=2,2'-bipyridine (bpy), 2,2'-bipyrimidine (bpm),1,10-phenanthroline (phen), pyrazino[2,3-f]quinoxaline (tap), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), dipyrido[2,3-a:2',3'-c]phenazine (dppz)] 2-7 have been prepared in a stepwise manner by treatment of RhCl(3).3H(2)O with the appropriate polypyridyl ligand (pp) followed by 1,4,7-trithiacyclononane. Interactions of the polypyridyl complexes with DNA were investigated by CD and UV/visible spectroscopy and by gel electrophoresis. The dpq complex 6 cleaves DNA exiguously in the dark, but UV irradiation is required to induce nuclease activity for the bpy complex 2. Whereas 2 [IC(50) values: 12.8 (+/-0.2) and 4.4 (+/-0.1)microM] exhibits significantly higher cytotoxicities towards MCF-7 and HT-29 cells than 4 [IC(50) values: 36.3 (+/-6.0) and 72.2 (+/-8.0)], the activity of complexes in the series 4/6/7 correlates directly with the size of the polypyridyl ligand, as documented by their respective IC(50) values of 72.2 (+/-8.0), 20.9 (+/-2.8) and 7.4 (+/-2.2) towards HT-29 cells. Complexes of the nitrogen-rich ligands bpm (3) [IC(50) values: 1.7 (+/-0.5) and 1.9 (+/-0.1)microM] and tap (5) [IC(50) values: 11.5 (+/-0.6) and 7.6 (+/-4.8)microM] are considerably more potent than their bpy and phen counterparts 2 and 4. Measurement of the lactate dehydrogenase release for lymphoma (BJAB) cells after 1h incubation demonstrates that unspecific necrosis is negligible for the most active compounds 3 and 7. Specific cell death apoptosis via DNA fragmentation was detected for BJAB cells after 72h incubation and significant loss of the mitochondrial membrane potential in lymphoma cells indicates that the intrinsic pathway is involved. Show less

Mitochondrial dysfunction, which is closely related to intracellular calcium overload and excessive free radicals, is an important cause of Alzheimer's disease (AD). However, molecular mechanisms of the mitochondrial Ca(2+) disregulation induced by oxidative stress in AD are still obscure. In an effort to gain a further understanding of this problem, we investigated the effects of superoxide anion, a primary free radical, on the expression of uncoupling proteins (UCPs) and the mitochondrial free Ca(2+) levels in the neuroblastoma SH-SY5Y cell line (neo) and stably expressed wild-type human APP(APP) and APP-Swedish mutation (APPsw) SH-SY5Y cells. It was found that UCP2 and UCP4 protein levels were upregulated in neo but downregulated in APP and APPsw cells by the superoxide anion. Our results show that the superoxide anion can regulate protein levels of UCP2 and UCP4 in SH-SY5Y cells, and the mitochondrial free Ca(2+) shifted their levels, tightly coupled with the protein levels of UCPs. When UCP2 and UCP4 were knocked down by siRNA, the result was reversed. These data suggest that the superoxide anion can regulate the mitochondrial free Ca(2+) by regulating the expression of UCPs. These observations also indicate that UCPs can be potential targets in pathotherapy prevention of AD. Show less

Cisplatin, carboplatin, and oxaliplatin are three FDA-approved members of the platinum anticancer drug family. These compounds induce apoptosis in tumor cells by binding to nuclear DNA, forming a variety of structural adducts and triggering cellular responses, one of which is the inhibition of transcription. In this report we present (i) a detailed review of the structural investigations of various Pt-DNA adducts and the effects of these lesions on global DNA geometry; (ii) research detailing inhibition of cellular transcription by Pt-DNA adducts; and (iii) a mechanistic analysis of how DNA structural distortions induced by platinum damage may inhibit RNA synthesis in vivo. A thorough understanding of the molecular mechanism of action of platinum antitumor agents will aid in the development of new compounds in the family. Show less

BACKGROUND: Cisplatin has been in use for 40 years for treatment of germ line and other forms of cancer. Oxaliplatin is approved for treatment of metastatic colorectal cancer. Thirty to forty percent of cancer patients receiving these agents develop pain and sensory loss. Oxaliplatin induces distinctive cold-associated dysesthesias in up to 80% of patients. RESULTS: We have established mouse models of cisplatin and oxaliplatin-induced neuropathy using doses similar to those used in patients. Adult male C57BL6J mice were treated with daily intraperitoneal injection for 5 days, followed by 5 days of rest, for two cycles. Total cumulative doses of 23 mg/kg cisplatin and 30 mg/kg oxaliplatin were used. Behavioral evaluations included cold plate, von Frey, radiant heat, tail immersion, grip strength and exploratory behavior at baseline and at weekly intervals for 8 weeks. Following two treatment cycles, mice in the cisplatin and oxaliplatin treatment groups demonstrated significant mechanical allodynia compared to control mice. In addition, the cisplatin group exhibited significant thermal hyperalgesia in hind paws and tail, and the oxaliplatin group developed significant cold hyperalgesia in hind paws. CONCLUSION: We have therefore established a model of platinum drug-induced painful peripheral neuropathy that reflects the differences in early thermal pain responses that are observed in patients treated with either cisplatin or oxaliplatin. This model should be useful in studying the molecular basis for these different pain responses and in designing protective therapeutic strategies. Show less

AbstractWe describe a scoring and modeling procedure for docking ligands into protein models that have either modeled or flexible side‐chain conformations. Our methodical contribution comprises a procedure for generating new potentials of mean force for the ROTA scoring function which we have introduced previously for optimizing side‐chain conformations with the tool IRECS. The ROTA potentials are specially trained to tolerate small‐scale positional errors of atoms that are characteristic of (i) side‐chain conformations that are modeled using a sparse rotamer library and (ii) ligand conformations that are generated using a docking program. We generated both rigid and flexible protein models with our side‐chain prediction tool IRECS and docked ligands to proteins using the scoring function ROTA and the docking programs FlexX (for rigid side chains) and FlexE (for flexible side chains). We validated our approach on the forty screening targets of the DUD database. The validation shows that the ROTA potentials are especially well suited for estimating the binding affinity of ligands to proteins. The results also show that our procedure can compensate for the performance decrease in screening that occurs when using protein models with side chains modeled with a rotamer library instead of using X‐ray structures. The average runtime per ligand of our method is 168 seconds on an Opteron V20z, which is fast enough to allow virtual screening of compound libraries for drug candidates. Proteins 2009. © 2008 Wiley‐Liss, Inc. Show less

We present here molecular dynamics simulations and DNA conformational dynamics for a series of trinuclear platinum [Pt(3)(HPTAB)](6+)-DNA adducts [HPTAB = N,N,N',N',N'',N''-hexakis (2-pyridyl-methyl)-1,3,5-tris(aminomethyl) benzene], including three types of bifunctional crosslinks and four types of trifunctional crosslinks. Our simulation results reveal that binding of the trinuclear platinum compound to a DNA duplex induces the duplex unwinding in the vicinity of the platination sites, and causes the DNA to bend toward the major groove. As a consequence, this produces a DNA molecule whose minor groove is more widened and shallow compared to that of an undamaged bare-DNA molecule. Notably, for trifunctional crosslinks, we have observed extensive DNA conformational distortions, which is rarely seen for normal platinum-DNA adducts. Our findings, in this study, thus provide further support for the idea that platinum compounds with trifunctional intra-strand or long-range-inter-strand cross-linking modes can generate larger DNA conformational distortions than other types of cross-linking modes. Show less

Chlorido osmium(II) arene [(eta(6)-biphenyl)Os(II)(X-pico)Cl] complexes containing X = Br (1), OH (2), and Me (3) as ortho, or X = Cl (4), CO(2)H (5), and Me (6) as para substituents on the picolinate (pico) ring have been synthesized and characterized. The X-ray crystal structures of 1 and 6 show typical "piano-stool" geometry with intermolecular pi-pi stacking of the biphenyl outer rings of 6. At 288 K the hydrolysis rates follow the order 2 >> 6 > 4 > 3 > 5 >> 1 with half-lives ranging from minutes to 4.4 h illustrating the influence of both electronic and steric effects of the substituents. The pK(a) values of the aqua adducts 3A, 4A, 5A, and 6A were all in the range of 6.3-6.6. The para-substituted pico complexes 4-6 readily formed adducts with both 9-ethyl guanine (9EtG) and 9-ethyl adenine (9EtA), but these were less favored for the ortho-substituted complexes 1 and 3 showing little reaction with 9EtG and 9EtA, respectively. Density-functional theory calculations confirmed the observed preferences for nucleobase binding for complex 1. In cytotoxicity assays with A2780, cisplatin-resistant A2780cis human ovarian, A549 human lung, and HCT116 colon cancer cells, only complexes 4 (p-Cl) and 6 (p-Me) exhibited significant activity (IC(50) values < 25 microM). Both of these complexes were as active as cisplatin in A2780 (ovarian) and HCT116 (colon) cell lines, and even overcome cisplatin resistance in the A2780cis (ovarian) cell line. The inactivity of 5 is attributed to the negative charge on its para carboxylate substituent. These data illustrate how the chemical reactivity and cancer cell cytotoxicity of osmium arene complexes can be controlled and "fine-tuned" by the use of steric and electronic effects of substituents on a chelating ligand to give osmium(II) arene complexes which are as active as cisplatin but have a different mechanism of action. Show less

The intrinsic factor (IF) vitamin B(12) ileum anchored receptor, cubilin, mediates endocytotic uptake of the IF complex of vitamin B(12) to the blood serum. This receptor was targeted for the selective delivery and accumulation of a new bioprobe, a B(12) conjugate of rhenium 2, in the cubilin expressing placental choriocarcinoma BeWo cell line. Competitive uptake and cytotoxicity assays of 2 were investigated and interactions with nuclear DNA explored. In addition, the mechanism of internalization of 2 was confirmed to proceed in an IF-cubilin mediated fashion via siRNA transfection experiments. These studies show the great potential of cubilin as a new target for the delivery of B(12) based conjugates for cancer diagnostics and/or treatment. Show less

Lithiation of 1-benzyl-1H-tetrazole followed by transmetallation with [AuCl(PPh3)], [Au(C6F5)(tht)] or [AuCl(tht)] (tht = tetrahydrothiophene) and subsequent alkylation afforded cationic 1-benzyl-4-methyl-4,5-dihydro-1H-1,2,3,4-tetrazol-5-ylidene(triphenylphosphine)gold(I), 1, neutral 1-benzyl-4-methyl-4,5-dihydro-1H-1,2,3,4-tetrazol-5-ylidene(pentafluorophenyl)gold(I), 2, and a cationic biscarbene complex, bis(1-benzyl-4-methyl-4,5-dihydro-1H-1,2,3,4-tetrazol-5-ylidene)gold(I), 3. The first complex underwent a homoleptic rearrangement in solution to form 3. Reaction of [Au(N3)PPh3] with the three isocyanides (CH3)2C6H3NC, tBuNC and CyNC, respectively, yielded the corresponding neutral tetrazolyl(phosphine) complexes of gold, [1-(2,6-dimethylphenyl)-1H-tetrazol-5-yl](triphenylphosphine)gold(I), 4, [1-(tert-butyl)-1H-tetrazol-5-yl](triphenylphosphine)gold(I), 6, and [1-(cyclohexyl)-1H-tetrazol-5-yl](triphenylphosphine)gold(I), 7. Alkylation of 4 with methyl triflate on N4 allowed isolation of the crystalline carbene complex 1-(2,6-dimethylphenyl)-4-methyl-4,5-dihydro-1H-1,2,3,4-tetrazol-5-ylidene)(triphenylphosphine)gold(I), 5. Complex 7 was not isolable in pure form but converts by isocyanide substitution of triphenylphosphine into [1-cyclohexylisocyanide][1-(cyclohexyl)-1H-tetrazol-5-yl]gold(I), 8. From a product mixture of 7 and 8 the transformed molecules [(cyclohexylamino)(ethoxy)carbene](1-cyclohexyl-1H-tetrazol-5-yl)gold(I), 9, and [bis(cyclohexylamino)carbene](1-cyclohexyltetrazol-5-yl)gold(I), 10, co-crystallised spontaneously after a long time at −20 °C. Show less

AbstractRare Earth Halides Ln4X5Z. Part 2: An Orthorhombic Variant of Ln4X5ZThe compounds Ln4Cl5C2 (Ln = La, Pr) and the isotypic bromides of lanthanum and cerium are prepared by reaction of LnX3, Ln metal and carbon in sealed Ta‐ampoules at temperature 900 °C < T < 950 °C. They crystallize in the orthorhombic space group Immm: La4Cl5C2: a = 3.9208(8) Å, b = 7.9446(16) Å, c = 17.2967(35) Å, Pr4Cl5C2: a = 3.8475(8) Å, b = 7.7590(16) Å, c = 17.0105(34) Å, La4Br5C2: a = 3.9950(8) Å, b = 8.2770(17) Å, c = 18.1010(36) Å and Ce4Br5C2: a = 3.9835(8) Å, b = 8.1865(16) Å, c = 18.0171(36) Å.In the crystal structure the metal atoms are connected to Ln6 octahedra centered by C2 units. The Ln6C octahedra are condensed via opposite egdes to chains and connected via X atoms. The special interconnection of the M6X12C2 chains by the halogen atoms, Ln2n+2Xi−i2/2Xi−i(2n−2)/2Xi−a4/2Xa−i4/2 (n = 1), results in an orthorhombic crystal structure in contrast to the monoclinic variant described in part 1. The C2 groups together with the halogen atoms form ccp anionic substructure, and 4/5 of the octahedral voids is occupied by the Ln atoms. The C‐C distances vary between 1.41 und 1.44Å corresponding to a shortened C–C single bond and a lengthened C=C double bond. For metal‐metal bonds are 1 < e− < 3 electrons available suggesting an ionic formulation (Ln3+)4(X−)5C2m− e− with C26− or C24− ions (4 < m < 6). Show less

This work is the first in-depth study of osmium binding to DNA and confirms the pharmacological activity of a new class of anticancer metallodrugs. We investigated the interactions between the potential biological target DNA and four osmium(II) arene complexes, of the type [(eta 6-arene)Os(LL)Cl]n+, where arene = biphenyl or p-cymene and LL = ethylenediamine, picolinate, or oxinate in an effort to understand their mechanism of action. Most notably we show that these complexes bind to DNA. DNA adducts of the OsII complexes that exhibit promising cytotoxic effects in ovarian tumor cell lines largely distort its conformation. The data are consistent with DNA binding of the complexes containing biphenyl as the arene ligand that involves combined coordination to guanine residues and noncovalent interactions between the arene ligand and DNA. The results also indicate both a mechanism of action and a detoxification mechanism for OsII arene compounds different from those of cisplatin. Show less

The synthesis, characterization, in vitro imaging, and cytotoxic properties of a new folate conjugate of rhenium(I) are reported. The conjugate [FA-PEG-BQAV-Re(CO)3]+ (gamma-4) was screened against an adriamycin- and cisplatin-resistant human ovarian cancer cell line (A2780/AD) that overexpresses the folate receptor (FR). Compound gamma-4 was internalized by a folate-receptor-mediated endocytotic pathway, which results in internal accumulation of gamma-4. This was contrasted with a FR-negative Chinese hamster ovary cell line in which no internalization of gamma-4 was observed. gamma-4 was found to be cytotoxic with IC(50) values of 189 and 78 microM at 6 and 24 h, respectively, toward the FR-positive cell line. This is in contrast to the IC(50) value of 502 microM at 6 h and 84 microM at 24 h for cisplatin in the same cell line, with a significantly greater toxicity at the earlier time point. The cytotoxicity of gamma-4 as explained by interactions that occur between the rhenium(I) complex moiety and DNA is described. Show less

Reactive oxygen species (ROS) are considered a key factor in the heart aging process. Mitochondrial respiration is an important site of ROS generation and a potential contributor to heart functional changes with aging. We have examined the effects of aging on various parameters related to mitochondrial bioenergetics in rat heart, such as complex I activity, oxygen consumption, membrane potential, ROS production, and cardiolipin content and oxidation. A loss in complex I activity, state 3 respiration, and membrane potential was found in mitochondria with aging. The capacity of mitochondria to produce H(2)O(2) was significantly increased in aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, significantly decreased as a function of aging, whereas there was a significant increase in the level of oxidized cardiolipin. The lower complex I activity in mitochondria from aged rats could be almost completely restored to the level of young heart by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that aging causes heart mitochondrial complex I deficiency, which can be attributed to ROS-induced cardiolipin peroxidation. These results may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with heart aging. Show less