Nonionic but water soluble, [Glycine-Pd-Alanine] and [Glycine-Pd-Valine] complexes. Their synthesis,characterization, antitumor activities and rich DNA/HSA interaction studies

Two novel, neutral and water soluble Pd(II) complexes of formula [Pd(Gly)(Ala)] (1) and [Pd(Gly)(Val)] (2) (Gly, Ala, and Val are anionic forms of glycine, alanine, and valine amino acids, respectively) have been synthesized and characterized by FT-IR, UV–Vis, ¹H-NMR, elemental analysis, and molar conductivity measurement. The data revealed that each amino acid binds to Pd(II) through the nitrogen of –NH₂ and the oxygen of –COO^– groups and acts as a bidentate chelate. These complexes have been assayed against leukemia cells (K₅₆₂) using MTT method. The results indicated that both of the complexes display more cytotoxicity than the well-known anticancer drug, cisplatin. The interaction of the compounds with calf thymus DNA (CT-DNA) and human serum albumin (HSA) were assayed by a series of experimental techniques including electronic absorption, fluorescence, viscometry, gel electrophoresis, and FT-IR. The results indicated that the two complexes have interesting binding propensities toward CT-DNA as well as HSA and the binding affinity of (1) is more than (2). The fluorescence data indicated that both complexes strongly quench the fluorescence of ethidium bromide–DNA system as well as the intrinsic fluorescence of HSA via static quenching procedures. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) calculated from the fluorescence studies showed that hydrogen bonds and van der Waals interactions play a major role in the binding of the complexes to DNA and HSA. We suggest that both of the Pd(II) complexes exhibit the groove binding mode with CT-DNA and interact with the main binding pocket of HSA.

Communicated by Ramaswamy H. Sarma     

Investigations into some aryl substituted bis(thiosemicarbazones) and their copper complexes

The synthesis of three bis(thiosemicarbazone) compounds formed by the reaction of benzil with either thiosemicarbazide, 4-methyl-3-thiosemicarbazide or 4-phenyl-3-thiosemicarbazide are reported. The compounds were characterised by NMR spectroscopy, mass spectrometry and in the case of benzil bis(4-methyl-3-thiosemicarbazone) and benzil bis(4-phenyl-3-thiosemicarbazone) by X-ray crystallography. Attempts to purify benzil bis(thiosemicarbazone) and benzil bis (4-methyl-3-thiosemicarbazone) by recrystallisation resulted in the isolation of cyclised products that were characterised by X-ray crystallography. The 3 bis(thiosemicarbazone) compounds were used to synthesise both Cu(II) and Cu(I) complexes. The copper(II) complexes were formed by the reaction of the proligands with copper(II) acetate which gave neutral copper(II) complexes in which the thiosemicarbazone is doubly deprotonated, acting as a dianionic ligand. The copper(II)-benzil bis(4-phenyl-3-thiosemicarbazonato) complex was characterised by X-ray crystallography to show the copper in an essentially square planar N₂S₂ environment. The copper(I) complexes were synthesised by reacting the bis (thiosemicarbazone) ligands with [Cu(CH₃CN)₄]PF₆ to give cationic complexes. The copper(I)-benzil-bis(thiosemicarbazone) complex was characterised by X-ray crystallography which revealed that the complex was a dimeric dication. Each of the benzil bis(thiosemicarbazone) ligands act as a bidentate N,S donor to each copper(I) atom, forming an overall helical structure in which each copper atom is in a strongly distorted tetrahedral N₂S₂ environment. Electrochemical measurements show that the copper(II)-benzil bis(thiosemicarbazonato) complex undergoes a reversible reduction at biologically accessible potentials.     

Synthesis,characterization, DNA and HSA binding studies of isomeric Pd (II) antitumor complexes using spectrophotometry techniques

Two new Palladium(II) isomeric complexes, [Pd (Gly)(Leu)](I) and [Pd (Gly)(Ile)](II), where Gly is glycine, and Leu and Ile are isomeric amino acids (leucine and isoleucine), have been synthesized and characterized by elemental analysis, molar conductivity measurements, FT-IR, ¹H NMR, and UV–Vis. The complexes have been tested for their In vitro cytotoxicity against cancer cell line K₅₆₂ and their binding properties to calf thymus DNA (CT-DNA) and human serum albumin (HSA) have also been investigated by multispectroscopic techniques. Interactions of these complexes with CT-DNA were monitored using gel electrophoresis. The energy transfer from HSA to these complexes and the binding distance between HSA and the complexes (r) were calculated. The results obtained from these studies indicated that at very low concentrations, both complexes effectively interact with CT-DNA and HSA. Fluorescence studies revealed that the complexes strongly quench DNA bound ethidium bromide as well as the intrinsic fluorescence of HSA through the static quenching procedures. Binding constant (K_b), apparent biomolecular quenching constant (k_q), and number of binding sites (n) for CT-DNA and HSA were calculated using Stern–Volmer equation. The calculated thermodynamic parameters indicated that the hydrogen binding and vander Waals forces might play a major role in the interaction of these complexes with HSA and DNA. Thus, we propose that the complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of I > II with DNA- and II > I with HSA-binding.     

Multispectroscopic insight,morphological analysis and molecular docking studies of CuII-based chemotherapeutic drug entity with human serum albumin (HSA) and bovine serum albumin (BSA)

The interaction studies of Cu^II nalidixic acid–DACH chemotherapeutic drug entity, [C₃₆H₅₀N₈O₆Cu] with serum albumin proteins, viz., human serum albumin (HSA) and bovine serum albumin (BSA) employing UV–vis, fluorescence, CD, FTIR and molecular docking techniques have been carried out. Complex [C₃₆H₅₀N₈O₆Cu] demonstrated strong binding affinity towards serum albumin proteins via hydrophobic contacts with binding constants, K?=?3.18?×?10⁵ and 7.44?×?10⁴ M^–1 for HSA and BSA, respectively implicating a higher binding affinity for HSA. The thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were also calculated and the interaction of complex [C₃₆H₅₀N₈O₆Cu] with HSA and BSA was found to be enthalpy and entropy favoured, nevertheless, complex [C₃₆H₅₀N₈O₆Cu] demonstrated higher binding affinity towards HSA than BSA evidenced from its higher binding constant values. Time resolved fluorescence spectroscopy (TRFS) was carried out to validate the static quenching mechanism of HSA/BSA fluorescence. The collaborative results of spectroscopic studies indicated that the microenvironment and the conformation of HSA and BSA (α–helix) were significantly perturbed upon interaction with complex [C₃₆H₅₀N₈O₆Cu]. Hirshfeld surfaces analysis and fingerprint plots revealed various intermolecular interactions viz., N–H····O, O–H····O and C–H····O linkages in a 2–dimensional framework that provide crucial information about the supramolecular architectures in the complex. Molecular docking studies were carried out to ascertain the preferential binding mode and affinity of complex [C₃₆H₅₀N₈O₆Cu] at the target site of HSA and BSA. Furthermore, only for Transmission electroscopy microscopy micrographs of HSA and BSA in presence of complex [C₃₆H₅₀N₈O₆Cu] revealed major protein morphological transitions and aggregation which validates efficient delivery of complex by serum proteins to the target site.

Communicated by Ramaswamy H. Sarma     

Biological evaluations of newly-designed Pt(II) and Pd(II) complexes using spectroscopic and molecular docking approaches

To perform biological evaluations of newly-designed Pt(II) and Pd(II) complexes, the present study was conducted with targeted protein human serum albumin (HSA) and HCT116 cell line as model of human colorectal carcinoma. The binding of Pt(II) and Pd(II) complexes to HSA was analyzed using fluorescence spectroscopy and molecular docking. The thermal stability and alterations in the secondary structure of HSA in the presence of Pt(II) and Pd(II) complexes were investigated using the thermal denaturation method and circular dichroism (CD) spectroscopy. The cytotoxicity of the Pt(II) and Pd(II) complexes was studied against the HCT116 cell line using MTT assay. The binding analysis revealed that the fluorescence findings were well in agreement with docking results such that there is only one binding site for each complex on HSA. Binding constants of 8.7?×?10³ M^?1, 2.65?×?10³ M^?1, 0.3?×?10³ M^?1, and 4.4?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 25?°C, respectively. Also, binding constants of 1.9?×?10³ M^?1, 15.17?×?10³ M^?1, 1.9?×?10³ M^?1, and 13.1?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 37?°C, respectively. The results of CD and thermal denaturation showed that the molecular structure of HSA affected by interaction with Pt(II) and Pd(II) complexes is stable. Cytotoxicity studies represented the growth suppression effect of the Pt(II) and Pd(II) complexes toward the human colorectal carcinoma cell line. Therefore, the results suggest that the new designed Pt(II) and Pd(II) complexes are well promising candidates for use in cancer treatment, particularly for human colorectal cancer.

Communicated by Ramaswamy H. Sarma     

New multinuclear Scaffold molybdocene-gold lidocaine complex: DNA/HSA binding,molecular docking,cytotoxicity and mechanistic insights

The new heteronuclear molybdocene-gold complex 1, [(η⁵-Cp)₂Mo^II[(μ²-η²-dtc)₂Nap]Au^III(LC)](PF₆), (η⁵-Cp: η⁵-cyclopentadienyl, (dtc)₂Nap: 2,7-bis(dithiocarbamate)naphthalene, LC: lidocaine) was synthesized and evaluated for biological activity. With the aim of assessing the possible DNA-binding mode, the interaction of the complex 1 with calf thymus DNA (CT DNA) was investigated by UV spectroscopy, emission titration, and viscosity measurement. Also, the binding of the complex to human serum albumin (HSA) was considered by UV–Vis and fluorescence emission spectroscopy. Moreover, molecular docking was used for modeling of the binding of the complex to DNA and HSA. These experimental results were confirmed by the results of molecular docking concerning the lowest binding energy. The cytotoxicity of the heterometallic complex 1 has been evaluated against a panel of several cancer cell lines with low micromolar IC₅₀ (72?h) values, according to its cellular uptake and also versus HEK293 nonmalignant fibroblasts. Moreover, the complex 1 showed the induction of apoptotic process.

Communicated by Ramaswamy H. Sarma     

Study on the interaction of fisetholz with BSA/HSA by multi-spectroscopic,cyclic voltammetric,and molecular docking technique

The interaction of fisetholz with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated by multi-spectroscopic, cyclic voltammetric, and molecular docking technique. The results revealed that there was a static quenching of BSA/HSA induced by fisetholz. The binding constants (K_a) and binding sites (n) were calculated at different temperatures (293, 303, and 311?K). The enthalpy change (ΔH) were calculated to be –17.20?kJ mol^?1 (BSA) and –18.28?kJ mol^?1 (HSA) and the entropy change (ΔS) were calculated to be 35.41?J mol^?1 (BSA) and 24.02?J mol^?1 (HSA), respectively, which indicated that the interaction between fisetholz and BSA/HSA was mainly by electrostatic attraction. Based on displacement experiments using site probes, indomethacin and ibuprofen, the binding site of fisetholz to BSA/HSA was identified as sub-domain IIIA, which was further confirmed by molecular docking method. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺, and Fe³⁺ on fisetholz-BSA or fisetholz-HSA complex. The spectra of synchronous fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) all showed that fisetholz binding to BSA/HSA leads to secondary structures change of the two serum albumins. According to the Förster non-radiation energy transfer theory, the binding distance between fisetholz and BSA/HSA was 2.94/4.68?nm. The cyclic voltammetry as a supporting tool also indicated that fisetholz interacted with protein.

Communicated by Ramaswamy H. Sarma     

Lipoprotein-associated phospholipase A2, and subsequent cardiovascular events and mortality among patients with coronary heart disease

Objective: The objective of this study is to evaluate the relevance of Lp-PLA₂ to risk prediction among coronary heart disease (CHD) patients.

Methods: Lp-PLA₂ activity was measured in 2538 CHD patients included in the Bezafibrate Infarction Prevention (BIP) study.

Results: Adjusting for patient characteristics and traditional risk factors, 1 standard deviation of Lp-PLA₂ was associated with a hazard ratio (HR) of 1.12 (95% confidence interval (CI): 1.00–1.25) for mortality and 1.03 (0.93–1.14) for cardiovascular events. Lp-PLA₂ did not significantly improve model discrimination, or calibration nor result in noteworthy reclassification.

Conclusions: Our results do not support added value of Lp-PLA₂ for predicting cardiovascular events or mortality among CHD patients beyond traditional risk factor.     

Insights into the interaction of ulipristal acetate and human serum albumin using multi-spectroscopic methods,molecular docking,and dynamic simulation

Interaction between ulipristal acetate (UPA) and human serum albumin (HSA) was investigated in simulated physiological environment using multi-spectroscopic and computational methods. Fluorescence experiments showed that the quenching mechanism was static quenching, which was confirmed by the time-resolved fluorescence. Binding constants (K_a) were found to be 1?×?10⁵ L mol^?1, and fluorescence data showed one binding site. Thermodynamic constants suggested the binding process was mainly controlled by electrostatic interactions. Results from the competition experiments indicated that UPA bound to site I of HSA. Fourier transform infrared spectra, circular dichroism spectra, synchronous fluorescence spectra, and 3D fluorescence indicated that UPA can induce conformation change in the HSA. The content of α-helix and β-sheet increased, while β-turn decreased. Hydrophobicity around the tryptophan residues declined, whereas its polarity increased. Molecular docking results were consistent with the experimental results. Results suggested that UPA located at the hydrophobic cavity site I of HSA, and hydrophobic force played the key role in the binding process. Moreover, molecular dynamics simulation was performed to determine the stability of free HSA and HSA-UPA system. Results indicated that UPA can stabilize HSA to a certain degree and enhance the flexibility of residues around site I.

Communicated by Ramaswamy H. Sarma     

New binary copper(II) complexes containing intercalating ligands: DNA interactions,an unusual static quenching mechanism of BSA and cytotoxic activities

New binary copper(II) complexes – [Cu(4-mphen)₂(NO₃)]NO₃·H₂O (1), [Cu(5-mphen)₂ (NO₃)]NO₃·H₂O (2), the known complex [Cu(dmphen)₂(NO₃)]NO₃ (3) and [Cu(tmphen)₂ (NO₃)]NO₃·H₂O (4) - (4-mphen: 4-methyl-1,10-phenanthroline, 5-mphen: 5-methyl-1,10-phenanthroline, dmphen: 4,7-dimethyl-1,10-phenanthroline, tmphen: 3,4,7,8-tetramethyl-1,10-phenanthroline), have been synthesized and characterized by CHN analysis, ESI-MS, FTIR and single-crystal X-ray diffraction techniques. Interaction of these complexes with calf thymus DNA (CT-DNA) has been investigated by absorption spectral titration, ethidium bromide (EB) and Hoechst 33,258 displacement assay and thermal denaturation measurement. These complexes cleaved pUC19 plasmid DNA in the absence and presence of an external agent. Notably, in the presence of H₂O₂ as an activator, the cleavage abilities of these complexes are obviously enhanced at low concentration. Addition of hydroxyl radical scavengers like DMSO shows significant inhibition of the DNA cleavage activity of these complexes. BSA quenching mechanism was investigated with regard to the type of quenching, binding constant, number of binding locations and the thermodynamic parameters. The experimental results suggested that the probable quenching mechanism was an unusual static process and hydrophobic forces play a dominant role. The CT-DNA and BSA binding efficiencies of these complexes follow the order: 4 > 3 > 1 > 2. Furthermore, in vitro cytotoxicities of these complexes on tumor cells lines (Caco-2, MCF-7 and A549) and healthy cell line (BEAS-2B) showed that these complexes exhibited anticancer activity with low IC₅₀ values. The effect of hydrophobicity of the methyl-substituted phenanthrolines on DNA and protein binding activities of these complexes is discussed.     

Investigation on the interaction of newly designed potential antibacterial Zn(II) complexes with CT-DNA and HSA

Two Zn(II) complexes of formula [Zn(bpy)(Gly)]NO₃ (I) and [Zn(phen)(Gly)]NO₃ (II) (where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and Gly = glycine) were synthesized and characterized by elemental analysis, molar conductance measurements, UV–vis, FT-IR, and ¹H NMR spectra. The interaction ability of these complexes with calf thymus DNA was monitored using spectroscopic methods, including UV–vis absorption spectroscopy, ethidium bromide displacement, Fourier transform infrared, and electrophoretic mobility assay. Further, the human serum albumin interactions of complexes I and II were investigated using UV–vis absorption spectroscopy, fluorescence quenching, circular dichroism, and Fourier transform infrared. The results obtained from these analyses indicated that both complexes interact effectively with CT-DNA and HSA. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) at different temperatures were determined for CT-DNA and HSA. Also, the negative ΔH° and ΔS° values showed that both hydrogen bonds and van der Waals forces played major roles in the association of CT-DNA-Zn(II) and HSA-Zn(II) complex formation. The displacement experiments suggested that Zn(II)-complexes primarily bound to Sudlow’s site II of HSA. The distance between the donor (HSA) and the acceptor (Zn(II) complexes) was estimated on the basis of the Forster resonance energy transfer (FRET) and the alteration of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. The complexes follow the binding affinity order of I > II with DNA and II > I with HSA. Finally, Antibacterial activity of complexes I and II have been screened against gram positive and gram negative bacteria.     

Serum adenosine deaminase,catalase, and carbonic anhydrase activities in patients with renal cell carcinoma

Objectives: To determine whether serum levels of adenosine deaminase (AD), catalase (CAT), and carbonic anhydrase (CA) enzymes may be useful biomarkers in the diagnosis of renal tumors and may lead to early diagnosis of renal tumors.

Material and methods: The study included 33 patients with renal cell carcinoma (RCC) and 31 healthy controls. The activity of serum AD, CA, and CAT was determined and analyzed using the Giusti spectrophotometric method, H₂O₂ substrate, and C0₂ hydration, respectively.

Results: Serum AD and CA activity were significantly higher in patients with RCC than in controls. However, serum CAT activity was significantly lower in patients with RCC than in controls.

Conclusion: These markers might be potentially important as an additional biochemical tool for diagnosing RCC. We believe multidisciplinary studies are needed to plan patients’ preoperative and postoperative treatment and to create follow-up protocols.     

Biologically important hydrazide-containing fused azaisocytosines as antioxidant agents

Objectives: Two important classes of hydrazide-containing fused azaisocytosines were evaluated as possible antioxidants and characterised by UV spectroscopy.

Methods: 2,2-Diphenyl-1-picrylhydazyl (DPPH), nitric oxide (NO), hydrogen peroxide (H₂O₂) scavenging potencies and reducing power of molecules were evaluated.

Results: The strongest DPPH scavengers were found to be 9, showing the potency superior to that of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and comparable to that of ascorbic acid (AA), and 6, revealing the antioxidant potency superior to that of BHA, BHT, PG and Trolox. In turn, 3 and 9 were the most promising NO scavengers, exhibiting the potency superior to that of BHA, BHT (3 and 9) and AA (3). The most potent H₂O₂ scavengers proved to be 10 and 9 showing similar or even better neutralising potency than that of Trolox, BHT and BHA. Simultaneously, the majority of hydrazides revealed higher ferric reducing abilities than that of AA and BHT. Some structure-activity relationships were explored. A possible mechanism for the DPPH radical scavenging ability of hydrazide-containing molecules was proposed.

Discussion: Hydrazides 3, 6 and 9 with an antioxidant potential better or comparable to that of the well-known antioxidants are proposed as new antioxidant candidates.     

Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines

Context: Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen.

Objective: Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines.

Materials and methods: SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco’s modified Eagle’s medium for 5 days, and next cultured in Hypoxic Chamber in 1% O₂, 10% O₂, 21% O₂. Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software.

Results: We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line.

Conclusion: Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.     

Palladium(II) complexes of biorelevant ligands. Synthesis,structures, cytotoxicity and rich DNA/HSA interaction studies

In this work, a pair of new palladium(II) complexes, [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)], (where Gly is glycine, Phe is phenylalanine, and Tyr is tyrosine) were synthesized and characterized by UV–Vis, FT-IR, elemental analysis, ¹H-NMR, and conductivity measurements. The detailed ¹H NMR and infrared spectral studies of these Pd(II) complexes ascertain the mode of binding of amino acids to palladium through nitrogen of -NH₂ and oxygen of -COO^? groups as bidentate chelates. The Pd(II) complexes have been tested for in vitro cytotoxicity activities against cancer cell line of K₅₆₂. Interactions of these Pd(II) complexes with CT-DNA and human serum albumin were identified through absorption/emission titrations and gel electrophoresis which indicated significant binding proficiency. The binding distance (r) between these synthesized complexes and HSA based on Forster?s theory of non-radiation energy transfer were calculated. Alterations of HSA secondary structure induced by complexes were confirmed by FT-IR measurements. The results of emission quenching at three temperatures have revealed that the quenching mechanism of these Pd(II) complexes with CT-DNA and HSA were the static and dynamic quenching mechanism, respectively. Binding constants (K_b), binding site number (n), and the corresponding thermodynamic parameters were calculated and revealed that the hydrogen binding and hydrophobic forces played a major role when Pd(II) complexes interacted with DNA and HSA, respectively. We bid that [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)] complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of [Pd(Gly)(Tyr)] > [Pd(Gly)(Phe)] with CT-DNA- and HSA-binding.     

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability

Objectives: Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity.

Methods: In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H₂O₂-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H₂O₂-treated vector control and LCMT1-overexpressing cells.

Results: A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H₂O₂-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H₂O₂ treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton.

Discussion: These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.     

Vancomycin-sensitized photooxidation in the presence of the natural pigment vitamin B2: Interaction with excited states and photogenerated ROS

Sensitized photooxidation processes in the presence of natural pigments may provide an alternative to antibiotics degradation since these compounds are transparent to natural light irradiation, therefore, they can be degraded by the action of photosensitizers which absorb light and produce highly reactive species, especially those derived from molecular oxygen (ROS). Most antibiotics used currently belong to a group of pharmaceutical substances that have been considered a new type of contaminants due to their persistence and bioaccumulation in the environment.

Objective: In this context, we decided to investigate the kinetic and mechanistic aspects of Vancomycin (Vanco) photosensitized degradation in the presence of the natural pigment Riboflavin (Vitamin B2, Rf) and the artificial dye Rose Bengal (RB) for comparative purposes.

Methods: The study have been done by using Stationary photolysis, Laser flash photolysis, Time-resolved phosphorence detection of O₂(1Δg) experiments and Bactericidal activity evaluation. The experiments were carried out in aqueous solution at different pH values in order to establish relationships between the structure of the compound and its susceptibility to ROS-mediated photooxidation.

Results: Experimental evidence indicates that in the presence of Rf there is considerable contribution of the radical-mediated mechanism, while in the presence of RB the photooxidation process occurs exclusively through O₂(1Δg) and the reactivity to this excited species increases with increasing pH of the environment.

Discussion: The results obtained, have been shown that Rf can raise the photodegradation of Vanco by both the radical pathway and the O₂(¹Δ_g) mediated. Furthermore, the antibiotic is able to interact with the excited electronic states of Rf as well as O₂(¹Δ_g) generated by energy transfer between the excited triplet state of the photosensitizer and the oxygen ground state. The predominant mechanism for photodegradation of Vanco in the presence of the Rf is the radical via because of the considerable interaction with the excited triplet state of the photosensitizer demonstrated by laser flash photolysis experiments.

Microbiological test on Staphylococcus aureus ATCC25923 showed that the bactericidal activity of the antibiotic on the strain studied was affected by the sensitized photodegradation process, suggesting that photoproducts generated eventually do not retain the bactericidal properties of the original antibiotic.     

Metal complexes of naphthoquinone based ligand: synthesis,characterization, protein binding,DNA binding/cleavage and cytotoxicity studies

Protein binding, DNA binding/cleavage and in vitro cytotoxicity studies of 2-((3-(dimethylamino)propyl)amino)naphthalene-1,4-dione (L) and its four coordinated M(II) complexes [M(II) = Co(II), Cu(II), Ni(II) and Zn(II)] have been investigated using various spectral techniques. The structure of the ligand was confirmed by spectral and single crystal XRD studies. The geometry of the complexes has been established using analytical and spectral investigations. These complexes show good binding tendency to bovine serum albumin (BSA) exhibiting high binding constant values (10⁵ M^?1) when compared to free ligand. Fluorescence titration studies reveal that these compounds bind strongly with CT-DNA through intercalative mode (K_app 10⁵ M^?1) and follow the order: Cu(II) > Zn(II) > Ni(II) > Co(II) > L. Molecular docking study substantiate the strength and mode of binding of these compounds with DNA. All the complexes efficiently cleaved pUC18-DNA via hydroxyl radical mechanism and the Cu(II) complex degraded the DNA completely by converting supercoiled form to linear form. The complexes demonstrate a comparable in vitro cytotoxic activity against two human cancer cell lines (MCF-7 and A-549), which is comparable with that of cisplatin. AO/EB and DAPI staining studies suggest apoptotic mode of cell death, in these cancer cells, with the compounds under investigation.     

TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway

Rationale: Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored.

Objective: To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action.

Methods and Results: Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca²⁺]_i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca²⁺]_i and velocity of shortening as well as an acceleration in [Ca²⁺]_i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1^?/? mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14–22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca²⁺/calmodulin-dependent kinase II (CaMKII) (1–5 min) that correlated with increases in CM [Ca²⁺]_i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca²⁺]_i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L).

Conclusions: These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca²⁺ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.