Stabilization of Mn(II) and Mn(III) in mononuclear complexes derived from tridentate ligands with N2O donors: Synthesis, crystal structure, superoxide dismutase activity and DNA interaction studies

A new family of tridentate ligands PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)phenol), N-PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydrazono)methyl)napthalen-1-ol), Me-PhimpH (2-(1-(2-phenyl-2-(pyridine-2-yl)hydrazono)ethyl)phenol) have been synthesized and characterized. The ligands PhimpH and N-PhimpH after deprotonation react with manganese(II) and manganese(III) starting materials affording [Mn(Phimp)₂] (1), [Mn(Phimp)₂](ClO₄) (2), [Mn(N-Phimp)₂] (3), [Mn(N-Phimp)₂](ClO₄) (4). Complexes [Mn(Phimp)₂] (1) and [Mn(N-Phimp)₂] (3) convert to [Mn(Phimp)₂]⁺ (cation of 2) and [Mn(N-Phimp)₂]⁺ (cation of 4) respectively upon oxidation. Ligand Me-PhimpH stabilized only manganese(III) centre resulting [Mn(Me-Phimp)₂](ClO₄) (5). The molecular structures of [Mn(Phimp)₂], 1 and [Mn(Phimp)₂](ClO₄), 2 were determined by single crystal X-ray diffraction. X-ray crystal structures of 1 and 2 have revealed the presence of distorted octahedral MnN₄O₂ coordination sphere having meridionally spanning ligands. Electrochemical studies for the complexes showed Mn(II)/Mn(III), (E_1/2 = 0.14-0.40 V) and Mn(III)/Mn(IV), (E_1/2 = 0.80-1.06 V) couples vs. Ag/AgCl. The redox properties were exploited to examine superoxide dismutase (SOD) activity using Mn(II)/Mn(III) couple. The complexes 1, 2, 4 and 5 have been revealed to catalyze effectively the dismutation of superoxide () in xanthine-xanthine oxidase-nitro blue tetrazolium assay and IC₅₀ values were found to be 0.29, 0.39, 1.12 and 0.76 μM respectively. DNA interaction studies with complex 2 showed binding of DNA in a non-intercalative pathway. Complexes 1, 2 and 4 exhibited nuclease activity in presence of H₂O₂ and inhibition of activity was noted in presence of KI.     

Alpha-santalol,a chemopreventive agent against skin cancer,causes G<Subscript>2</Subscript>/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

Background

α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.

Methods

MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells.

Results

α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G₂/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G₂/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells.

Conclusions

This study for the first time identifies effects of α-santalol in G₂/M phase arrest and describes detailed mechanisms of G₂/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models.

     

Proteomics Characterization of Extracellular Space Components in the Human Aorta

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.Vascular cells, in particular vascular smooth muscle cells, produce and maintain a complex meshwork of ECM.¹ The ECM is not only the scaffold for the anchorage and mobility of residing cells but also absorbs and transduces the shear and strain forces of the blood flow. It is primarily composed of elastin, collagen, proteoglycans, and glycoproteins. The elastin fibers and type I and III fibrillar collagens form a rigid network of highly cross-linked interstitial matrix. They offer elasticity (elastin) and tensile strength (collagens). Proteoglycans, because of their negative charge, attract water and confer resistance to compression. Finally, glycoproteins participate in matrix organization and are essential for cell attachment.The vascular ECM also serves as a substrate for the binding and retention of secreted, soluble proteins of vascular cells as well as molecules coming from the circulation, including lipoproteins, growth factors, cytokines, proteases, and protease inhibitors. These components are invariably associated with ECM proteins, especially proteoglycans. Together they comprise the vascular extracellular environment and are pivotal for disease processes, such as atherosclerosis and aneurysm formation (1).Although proteomics has been previously applied to vascular tissues, only one study has specifically targeted the extracellular vascular environment (2). This study was focused on the isolation of intimal proteoglycans from human carotid arteries. Moreover, most proteomics studies use whole tissue lysates, which are rich in cellular proteins that inevitably mask the identification of the less abundant proteins of the vascular extracellular environment (3–5). Thus, the composition of the vascular ECM and its associated proteins remains poorly defined. In the present study, we used morphologically normal human aortic samples to develop a method for the extraction of proteins present in the extracellular environment, including ECM proteins and proteins attached to the ECM. We had three specific aims: first, to reduce the contamination with cellular proteins, thereby increasing the chance of identifying scarce extracellular proteins; second, to efficiently solubilize and deglycosylate ECM proteins to improve their analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS); and third, to interface the nanoflow LC system to a recently developed injection device, which splits the flow from the analytical column, to allow the reanalysis of the same sample during a single LC-MS/MS run (RePlay, Advion).Our methodology provides a detailed overview of the aortic ECM and its associated proteins, many reported for the first time in proteomics analysis of the vasculature. Most importantly, this method could be adapted for use with other tissues to further our understanding of the composition of extracellular environment and ECM turnover under various disease conditions.     

Electrochemical biosensor for catechol using agarose-guar gum entrapped tyrosinase

An electrochemical biosensor using tyrosinase was constructed for the determination of catechol. The enzyme was extracted from a plant source Amorphophallus companulatus and entrapped in agarose-guar gum composite biopolymer matrix. Catechol was determined by direct reduction of biocatalytically liberated quinone species at -0.1 V versus Ag/AgCl (3M KCl). The response was found to be linear and concentration dependent in the range of 6 x 10(-5) to 8 x 10(-4)M with a lower detection limit of 6 microM. It has reusability up to 20 cycles and a shelf life of more than 2 months when stored at 4 degrees C.     

Discovery and characterization of a novel inhibitor of matrix metalloprotease-13 that reduces cartilage damage in vivo without joint fibroplasia side effects

Matrix metalloproteinase-13 (MMP13) is a Zn(2+)-dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structure-based drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.     

A study of 7-deaza-2'-deoxyguanosine 2'-deoxycytidine base pairing in DNA

The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson–Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C–H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization.     

Contribution of dietary arginine to nitrogen utilisation and excretion in juvenile sea bass (Dicentrarchus labrax) fed diets differing in protein source

The role of dietary arginine in affecting nitrogen utilisation and excretion was studied in juvenile European sea bass (Dicentrarchus labrax) fed for 72 days with diets differing in protein sources (plant protein-based (PM) and fish-meal-based (FM)). Fish growth performance and nitrogen utilisation revealed that dietary Arg surplus was beneficial only in PM diets. Dietary Arg level significantly affected postprandial plasma urea concentrations. Hepatic arginase activity increased (P<0.05) in response to dietary Arg surplus in fish fed plant protein diets; conversely ornithine transcarbamylase activity was very low and inversely related to arginine intake. No hepatic carbamoyl phosphate synthetase III activity was detected. Dietary arginine levels did not affect glutamate dehydrogenase activity. A strong linear relationship was found between liver arginase activity and daily urea-N excretion. Dietary Arg excess reduced the proportion of total ammonia nitrogen excreted and increased the contribution of urea-N over the total N excretion irrespective of dietary protein source. Plasma and excretion data combined with enzyme activities suggest that dietary Arg degradation via hepatic arginase is a major pathway for ureagenesis and that ornithine-urea cycle is not completely functional in juvenile sea bass liver.