The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-beta (TGF-beta) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-beta-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.

Inhibition of Lactoperoxidase-Catalyzed Oxidation by Imidazole-Based Thiones and Selones: A Mechanistic Study

Herein, we describe the synthesis and biomimetic activity of a series of N,N-disubstituted thiones and selones that contain an imidazole pharmacophore. The N,N-disubstituted thiones do not show any inhibitory activity towards LPO-catalyzed oxidation reactions, but their corresponding N,N-disubstituted selones exhibit inhibitory activity towards LPO-catalyzed oxidation reactions. Substituents on the N atom of the imidazole ring appear to have a significant effect on the inhibition of LPO-catalyzed oxidation and iodination reactions. Selones 16, 17, and 19, which contain methyl, ethyl, and benzyl substituents, exhibit similar inhibition activities towards LPO-catalyzed oxidation reactions with IC50 values of 24.4, 22.5, and 22.5M, respectively. However, their activities are almost three-fold lower than that of the commonly used anti-thyroid drug methimazole (MMI). In contrast, selone 21, which contains a NCH2CH2OH substituent, exhibits high inhibitory activity, with an IC50 value of 7.2M, which is similar to that of MMI. The inhibitory activity of these selones towards LPO-catalyzed oxidation/iodination reactions is due to their ability to decrease the concentrations of the co-substrates (H2O2 and I2), either by catalytically reducing H2O2 (anti-oxidant activity) or by forming stable charge-transfer complexes with oxidized iodide species. The inhibition of LPO-catalyzed oxidation/iodination reactions by N,N-disubstituted selones can be reversed by increasing the concentration of H2O2. Interestingly, all of the N,N-disubstituted selones exhibit high anti-oxidant activities and their glutathione peroxidase (GPx)-like activity is 4-12-fold higher than that of the well-known GPx-mimic ebselen. These experimental and theoretical studies suggest that the selones exist as zwitterions, in which the imidazole ring contains a positive charge and the selenium atom carries a large negative charge. Therefore, the selenium moieties of these selones possess highly nucleophilic character. The 77SeNMR chemical shifts for the selones show large upfield shift, thus confirming the zwitterionic structure in solution.

An antioxidant nanozyme that uncovers the cytoprotective potential of vanadia nanowires

Nanomaterials with enzyme-like properties has attracted significant interest, although limited information is available on their biological activities in cells. Here we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme glutathione peroxidase by using cellular glutathione. Although bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origin (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson's and Alzheimer's disease.

Measurement of estrogen activity in two Chinese lakes

Estrogenic activities of samples from two Chinese lakes, Ya-Er Lake and Donghu Lake, were measured by in vitro recombinant yeast assay and found in both lakes polluted with industrial and domestic wastewater. In methanol extracts of lake water samples the estrogen-like activity was higher than in toluene extracts. These results have been inverted for solid samples. Furthermore, the EC50 value of the water samples was close to the original concentration in the lake.

Multifunctional G-Quadruplex Aptamers and Their Application to Protein Detection

Two significant G-quadruplex aptamers named AGRO100 and T30695 are identified as multi functional aptamers that can bind the protein ligands nucleolin or HIV-1 integrase and hemin. Besides their strong binding to target proteins, both AGRO100 and T30695 exhibit high hemin-binding affinities comparable to that of the known aptamer (termed PS2M) selected by the in vitro evolution process. Most importantly, their corresponding hemin-DNA complexes reveal excellent peroxidase-like activities. higher than that of the reported hemin-PS2M DNAzyme. This enables these multifunctional aptamers to be applied to the sensitive detection of proteins. which is demonstrated by applying AGRO100 to the chemiluminescence detection of nucleolin expressed at the surface of HeLa cells. Based on the specific AGRO100-nucleolin interaction, the surface-expressed nucleolin of HeLa cells is labeled in situ with the hemin-AGRO100 DNAzyme, and then determined in the luminol-H2O2 system.

G-quadruplex-based DNAzyme for sensitive mercury detection with the naked eye

Hg2+ is able to inhibit the peroxidase-like DNAzyme function of a T-containing G-quadruplex DNA via Hg2+-mediated T-T base pairs, which enables the visual detection of Hg2+ in the TMB-H2O2 reaction system with high selectivity and sensitivity.

Label-Free Colorimetric Detection of Aqueous Mercury Ion (Hg2+) Using Hg2+-Modulated G-Quadruplex-Based DNAzymes

Mercury ion (Hg2+) is able to specifically bind to the thymine-thymine (T-T) base pair in a DNA duplex, thus providing a rationale for DNA-based selective detection of Hg2+ with various means. In this work, we for the first time utilize the Hg2+-mediated T-T base pair to modulate the proper folding of G-quadruplex DNAs and inhibit the DNAzyme activity, thereby pioneering a facile approach to sense Hg2+ with colorimetry. Two bimolecular DNA G-quadruplexes containing many T residues are adopted here, which function well in low- and high-salt conditions, respectively. These G-quadruplex DNAs are able to bind hemin to form the peroxidase-like DNAzymes in the folded state. Upon addition of Hg2+, the proper folding of G-quadruplex DNAs is inhibited due to the formation of T-Hg2+-T complex. Ibis is reflected by the notable change of the Soret band of hemin when investigated by using UV-vis absorption spectroscopy. As a result of Hg2+ inhibition, a sharp decrease in the catalytic activity toward the H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) is observed, accompanied by a change in solution color. Through this approach, aqueous Hg2+ can be detected at 50 nM (10 ppb) with colorimetry in a facile way, with high selectivity against other metal ions.

COVALENT LIPOPROTEIN(A) ASSEMBLY: Characterization of Oxidase Activity Responsible for Catalyzing Covalent Lipoprotein(a) Assembly

Lipoprotein(a) (Lp(a)) has been identified as an emerging risk factor for the development of vascular diseases. The Lp(a) particle is assembled in a 2-step process upon secretion of the LDL and apo(a) components from hepatocytes. Work done by the Koschinsky group has identified an oxidase-like activity present in the conditioned medium (CM) harvested from human hepatoma (HepG2), as well as HEK 293 (human endothelian kidney) cells that catalyzes the rate of covalent Lp(a) formation. We have taken a candidate enzyme approach to identifying this oxidase activity. Specifically, we have proposed that the QSOX (Quiescin/sulfhydryl oxidase) is responsible for catalysis of covalent Lp(a) assembly. An oxidase activity assay developed by Dr. Thorpe (University of Delaware) was used to detect QSOX1 in CM harvested from cultured cell lines that catalyze covalent Lp(a) assembly. In addition, the QSOX1 transcript was identified in each cell line and quantified with the use of Real-Time RT-PCR. Quantitative assays of covalent Lp(a) assembly were performed to study some characteristics of the unkwown oxidase activity. First, conditioned medium was dialyzed through a 5 kDa cutoff, as this has previously been shown to reduce the aforementioned oxidase activity. Purified QSOX was then added back to the reaction and the rate of catalysis was observed. The addition of QSOX appeared to enhance the rate of covalent Lp(a) assembly in a dose-dependent manner. Additional covalent Lp(a) assembly assays were performed where various chemicals were added to determine whether Lp(a) assembly was affected. The addition of EDTA did not affect covalent assembly, suggesting that the oxidase activity may not be metallo-dependent. Moreover, dose-dependent addition of Calcium, DTT, Copper and glutathione to dialyzed medium also did not affect the rate of Lp(a) assembly. Taken together, these studies will aid in identifying the nature of the oxidase activity that catalyzes covalent Lp(a) assembly. This will provide us with valuable information on how Lp(a) particles are assembled, and may lead to the development of drugs inhibiting Lp(a) formation.

Synthesis, Kinetic Evaluation and Utilization of a Biotinylated Dipeptide Proline Diphenyl Phosphonate for the Disclosure of Dipeptidyl Peptidase IV-like Serine Proteases

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.

Discovery of Novel Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Reveals Chemical and Functional Diversity and In Vivo Activity in Rat Behavioral Models of Anxiolytic and Antipsychotic Activity

Modulators of metabotropic glutamate receptor subtype 5 (mGluR5) may provide novel treatments for multiple central nervous system (CNS) disorders, including anxiety and schizophrenia. Although compounds have been developed to better understand the physiological roles of mGluR5 and potential usefulness for the treatment of these disorders, there are limitations in the tools available, including poor selectivity, low potency, and limited solubility. To address these issues, we developed an innovative assay that allows simultaneous screening for mGluR5 agonists, antagonists, and potentiators. We identified multiple scaffolds that possess diverse modes of activity at mGluR5, including both positive and negative allosteric modulators (PAMs and NAMs, respectively). 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl) benzonitrile (VU0285683) was developed as a novel selective mGluR5 NAM with high affinity for the 2-methyl-6-(phenyl-ethynyl)-pyridine (MPEP) binding site. VU0285683 had anxiolytic-like activity in two rodent models for anxiety but did not potentiate phen-cyclidine-induced hyperlocomotor activity. (4-Hydroxypiperidin-1-yl)(4-phenylethynyl) phenyl) methanone (VU0092273) was identified as a novel mGluR5 PAM that also binds to the MPEP site. VU0092273 was chemically optimized to an orally active analog, N-cyclobutyl-6-((3-fluorophenyl) ethynyl) nicotinamide hydrochloride (VU0360172), which is selective for mGluR5. This novel mGluR5 PAM produced a dose-dependent reversal of amphetamine-induced hyperlocomotion, a rodent model predictive of antipsychotic activity. Discovery of structurally and functionally diverse allosteric modulators of mGluR5 that demonstrate in vivo efficacy in rodent models of anxiety and antipsychotic activity provide further support for the tremendous diversity of chemical scaffolds and modes of efficacy of mGluR5 ligands. In addition, these studies provide strong support for the hypothesis that multiple structurally distinct mGluR5 modulators have robust activity in animal models that predict efficacy in the treatment of CNS disorders.

Anticholinesterase-induced epileptiform activity in immature rat piriform cortex slices, in vitro

Purpose: Acute in vitro brain slice models are commonly used to study epileptiform seizure generation and to test anti-epileptic drug action. Seizure-like activity can be readily induced by manipulating external ionic concentrations or by adding convulsant agents to the bathing medium. We previously showed that epileptiform bursting was induced in slices of immature (P14–28) rat piriform cortex (PC) by applying oxotremorine-M, a potent muscarinic receptor agonist. Here, we examined whether raising levels of endogenous acetylcholine (ACh) by exposure to anticholinesterases, could also induce epileptiform events in immature (P12–14) or early postnatal (P7–9) rat PC brain slices. Methods: The effects of anticholinesterases were investigated in rat PC neurons using both extracellular MEA (P7–9 slices) and intracellular (P12–14 slices) recording methods. Results: In P7–9 slices, eserine (20 μM) or neostigmine (20 μM) induced low amplitude, low frequency bursting activity in all three PC cell layers (I–III), particularly layer III, where neuronal muscarinic responsiveness is known to predominate. In P12–14 neurons, neostigmine produced a slow depolarization together with an increase in input resistance and evoked cell firing. Depolarizing postsynaptic potentials evoked by intrinsic fibre stimulation were selectively depressed although spontaneous bursting was not observed. Neostigmine effects were blocked by atropine (1 μM), confirming their muscarinic nature. We conclude that elevation of endogenous ACh by anticholinesterases can induce bursting in early postnatal PC brain slices, further highlighting the epileptogenic capacity of this brain region. However, this tendency declines with further development, possibly as local inhibitory circuit mechanisms become more dominant.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the NO-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Employing immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-3H-Arginine to L-3H-Citrulline in a Ca2+/Calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform the activity of which is compromised in patients with coronary artery disease.

Antioxidant effect of functionalized alkyl-organotellurides: a study in vitro

We evaluated the in vitro antioxidant effect of alkyl-organotellurides A-D on lipid peroxidation and protein carbonylation in rat liver homogenates. The thiol oxidase and thiol peroxidase-like activities of compounds were investigated. delta-Aminolevulinic acid dehydratase (delta-ALA-D) activity was determined in rat liver homogenates. Compounds A-D protected against lipid peroxidation induced by Fe(2+)/EDTA and sodium nitroprusside (SNP). According to the confidence limits of the IC(50) values of compounds A-D, the IC(50) values for organotellurides followed the order: C (0.30 mu M) <= B (0.40 mu M) < D (0.68 mu M) < A (2.90 mu M), for Fe(2+)/EDTA, and B (0.21 mu M) <= C (0.33 mu M) < D (0.43 mu M) < A (1.21 mu M) for SNP-induced lipid peroxidation. Compounds A-D reduced protein carbonyl content to control levels. The results demonstrated an inverse correlation between thiol oxidase and delta-ALA-D activities. This study supports an antioxidant effect of organotellurides A-D on rat liver.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon citratus (lemongrass)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)