In vitro interactions of histones and α-crystallin

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone–α-crystallin binding with a K_d of 4 × 10^?7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone–α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone–α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.     

Oxidation of α-tocopherol in micelles and liposomes by the hydroxyl,perhydroxyl, and superoxide free radicals

Rates of oxidation of α-tocopherol by the hydroxyl- and superoxide free radicals were measured. The radicals were produced in known yields by radiolysis of aqueous solutions with gamma rays. Two main systems were used to dissolve the tocopherol; micelles, made up from charged and uncharged amphiphiles, and membranes made from dimyristyl phosphatidylcholine which could be charged by addition of stearyl amine or dicetyl phosphate. The HO. radicals were efficient oxidants of α-tocopherol in all systems, with up to 83% of radicals generated in micelle and 32% in membrane suspensions initiating the oxidation. The HO_{$\text{2}\text{?}$} radical was an even more effective oxidant, but when most of it was in the O form at neutral or alkaline pH, the oxidation rates became low. Tocopherol held in positively charged micelles or membranes was oxidized at a higher rate by the O than in uncharged or negative particles. Possible biological significance of these results is discussed.     

In vitro and in silico studies on membrane interactions of diverse Capsicum annuum flower γ-thionin peptides

Thionins are small, cysteine-rich peptides that play an important role in plant defense, primarily through their interactions with membranes. Eight novel γ-thionin peptides (CanThio1-8) were isolated from the flower of Capsicum annuum. Sequence analysis revealed that the peptides cluster into three groups. A representative peptide from each group (CanThio1, 2, and 3) was used for experimental characterization. Interestingly, peptides were found to possess some cytotoxic activity against normal human embryonic kidney cell line but higher cytotoxicity against cancer cell line MCF-7. CanThio3 peptide was chosen as a representative peptide to study the molecular mechanism of action on membranes. Microsecond timescale atomistic simulations of CanThio3 were performed in the presence of a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Simulations revealed that CanThio3 interacts with the bilayer and causes lipid thinning in the vicinity. Nonpolar amino acids specific to the α-core region of CanThio3 along with nonpolar residues in the γ-core region are seen to interact with the lipid tails. The differences in the amino acid sequence of CanThio peptides in these regions explain the variability in cytotoxic activities. In summary, our results demonstrate the membrane-mediated activity of a novel series of γ-thionin peptides from C. annuum.     

In vitro replication studies of carboxymethylated DNA lesions with Saccharomyces cerevisiae polymerase η

Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In this study, we examined how carboxymethylated DNA lesions, which included N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA), N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O(4)-carboxymethylthymidine (O(4)-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N(6)-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5'-nucleoside with high efficiency. By contrast, the polymerase could bypass N(4)-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O(4)-CMdT and N3-CMdT, and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors.     

In vitro anticancer studies of α- and β-d-glucopyranose betulin anomers

Four derivatives of betulin containing a d-glucopyranosyl moiety at C3 position were synthesized and characterized by ¹H and ¹³C NMR spectroscopy as well as mass spectrometry. The crystal structure of 28-O-acetylbetulin-3-yl-β-d-(2′,3′,4′,6′-tetra-O-acetyl)glucopyranoside was determined. The compounds were tested against fifteen tumor cell lines of different histogenic origins. The α- and β-anomers of 28-O-acetylbetulin-3-yl-d-glucopyranoside, exerted a dose dependent antiproliferative action towards the tumor cell lines. Treatment of HCT-116 cells for 24 h induced apoptosis, which was confirmed by the appearance of a typical ladder pattern in the DNA fragmentation assay and cell cycle analysis. The α- and β-anomers of 28-O-acetylbetulin-3-yl-d-glucopyranoside seem to induce apoptosis by activation of different upstream caspases on colon cancer HCT-116 cell line.     

Subcellular localization of proflavine derivative and induction of oxidative stress—In vitro studies

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem. 2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells.     

Synergic effects of NO and oxygen free radicals in the injury of ischemia-reperfused myocardium——ESR studies on NO free radicals generated from ischemia-reperfused myocardium

The ESR signal of NO bound to hemoglobin was detected during the ischemia-reperfusion of myocardium with low temperature ESR technique, and the synergic effects of NO and oxygen free radicals in the injury of the process were studied with this technique. Oxygen free radicals and NO bound to β-subunit of hemoglobin (β-NO complex) could be detected simultaneously in the ischemia-reperfused myocardium. Those signals could not be detected from the normal myocardium even in the presence of L-arginme. However, those signals could be detected and were dose-dependent with L-arginine in the ischemia-reperfused myocardiums and the signal could be suppressed with the inhibitor of NO synthetase, NG-nitro-L-arginine methylester (NAME). Measurement of the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart showed that L-arginine at lower concentration (<1 mmol/L) could protect the heart from the ischemia-reperfusion injury but at higher con     

In vitro radioprotection studies of organoselenium compounds: differences between mono- and diselenides

Organoselenium compounds belonging to the class of monoselenides, such as selenomethionine (SeM) and methylselenocysteine (MSeCys) and diselenides including selenocystine (SeCys) and selenopropionic acid (SePA), were examined for their comparative radioprotective effects using in vitro models. Effects of these compounds on the inhibition of γ-radiation induced lipid peroxidation in liposomes, protein carbonylation in bovine serum albumin (BSA) and strand breaks in pBR322 plasmid DNA, assessed, respectively, by the formation of thiobarbituric acid reactive substances, formation of 2,2′-dinitrophenyl hydrazine (DNPH) carbonyl complex and horizontal gel electrophoresis, were used to compare their radioprotective ability. The IC₅₀ values for SeCys, SePA, SeM and MSeCys for lipid peroxidation were 27 ± 1, 33 ± 2, 200 ± 8 and 163 ± 4 μM, respectively, and the values for inhibition of protein carbonylation were >200, 300 ± 6, 464 ± 8 and 436 ± 3 μM, respectively. Inhibition of DNA strand break formation was tested at 200 μM for all the compounds and SePA and SeCys exhibited a protective effect on DNA, while SeM and MSeCys did not lead to any protection. The in vitro cytotoxicity studies in normal and tumor cells revealed that MSeCys and SeM were not cytotoxic to lymphocytes and EL4 tumor cells at the concentrations employed. In contrast, SeCys was toxic, with a higher effect on tumor cells than lymphocytes. Our studies suggest that the non-toxic diselenides like SePA should be explored as protective agents against γ-irradiation induced damage.     

In vitro and in silico analysis of the Aspergillus nidulans DNA–CreA repressor interactions

The CreA protein mediates carbon catabolite repression in the fungus Aspergillus nidulans. Its DNA-binding domain belongs to the Cys₂-His₂ class, binding specifically to a 5′ SYGGRG 3′ nucleotide sequence. EMSA experiments showed that the CreA(G27D) mutation resulted in a 30-fold increase of the K_diss, and footprinting revealed a altered pattern of protein/DNA contacts. We modeled the CreA and the CreA(G27D) complexes in silico. A 15?ns molecular dynamics simulation of the solvated CreA(G27D) and CreA models was carried out using the MOE 2007.09 suite and the Amber99 force field. We have focused our analysis in residues Arg14, Glu16, His17, and Arg20 and Arg44, Asp46, and Arg50, previously, shown to be responsible for the specific contacts of the two Zn fingers. The electrostatic and the total potential energies showed the CreA(G27D) mutation to decrease the affinity of the complex, in agreement with the K_diss′s values. The in silico approach highlighted the role of the inter-finger linker. We identified several differential structural characteristics of the CreA and CreA(G27D)/DNA complexes and observed that the latter resulted in a lower dynamic flexibility of the complex.     

Synthesis of thiobarbituric acid derivatives: In vitro α-glucosidase inhibition and molecular docking studies

Synthesis, structure, and evaluation of in vitro α-glucosidase enzyme inhibition of a new class of diethylammonium salts of aryl substituted thiobarbituric acid is described. This protocol is straight, environmentally benign and efficient, involving Aldol-Michael addition reaction in one pot fashion. The 3D chemical structures of the synthesized compounds were assigned based on spectroscopic methods and X-ray single crystal diffraction analyses. All synthesized compounds 3a-3n were evaluated for their in vitro α-glucosidase enzyme inhibitory activity, whereas acarbose was used as the standard drug (IC₅₀ = 840 ± 1.73 µM). All tested compounds were found to possess varying degree of α-glucosidase enzyme inhibition activity with (IC₅₀ = 19.46 ± 1.84–415.8 ± 4.0 µM). Compound 3i (IC₅₀ = 19.4 ± 1.84 µM) exhibited the highest activity. To the best of knowledge this is the first report of the in vitro α-glucosidase enzyme inhibition by the diethylamonium salts of aryl substituted thiobarbituric acid. Furthermore, molecular docking studies of selected compounds were also performed to see interactions between active compounds and binding sites.     

Reaction mechanisms of DNT with hydroxyl radicals for advanced oxidation processes—a DFT study

In advanced oxidation processes (AOPs), the detailed degradation mechanisms of a typical explosive of 2,4-dinitrotoluene (DNT) can be investigated by the density function theory (DFT) method at the SMD/M062X/6-311+G(d) level. Several possible degradation routes for DNT were explored in the current study. The results show that, for oxidation of the methyl group, the dominant degradation mechanism of DNT by hydroxyl radicals (?OH) is a series of sequential H-abstraction reactions, and the intermediates obtained are in good agreement with experimental findings. The highest activation energy barrier is less than 20 kcal mol^?1. Other routes are dominated by an addition-elimination mechanism, which is also found in 2,4,6-trinitrotoluene, although the experiment did not find the corresponding products. In addition, we also eliminate several impossible mechanisms, such as dehydration, HNO₃ elimination, the simultaneous addition of two ?OH radials, and so on. The information gained about these degradation pathways is helpful in elucidating the detailed reaction mechanism between nitroaromatic explosives and hydroxyl radicals for AOPs.

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Graphical Abstract The degradation mechanism of an important explosive, 2,6-dinitrotoluene (DNT), by the hydroxyl radical for advanced oxidation progresses

     

In vivo interactions of TGF-β and extracellular matrix

TGF-β, a multifunctional cytokine, plays an important role in embryogenesis and in regulating repair and remodeling following tissue injury. Many of the biological actions of TGF-β are mediated by widespread effects on deposition of extracellular matrix. TGF-β stimulates the synthesis of individual matrix components including proteoglycans, collagens and glycoproteins. TGF-β also blocks matrix degradation by decreasing the synthesis of proteases and increasing the synthesis of protease inhibitors. Finally, TGF-β increases the synthesis of matrix receptors and alters their relative proportions on the surface of cells in a manner that could facilitate adhesion to matrix. All of these events have largely been demonstrated in vitro in cultured cells. In an experimental model of glomerulonephritis we have shown that TGF-β is responsible for the accumulation of pathological matrix in the glomeruli following immunological injury. Furthermore, all three of TGF-β's actions on extracellular matrix—increased synthesis, decreased degradation and modulation of receptors—have now been documented to be involved in matrix deposition in vivo in this model. Administration of the proteoglycan decorin suppressed TGF-β-induced matrix deposition in the nephritic glomeruli, thus confirming a physiological role for decorin as a regulator of TGF-β. Inhibitors of TGF-β may be important future drugs in treating fibrotic diseases caused by overproduction of TGF-β.     

Generation of superoxide radicals by copper–glutathione complexes: Redox-consequences associated with their interaction with reduced glutathione

The interaction between Cu²⁺ ions and GSH molecules leads to the swift formation of the physiologically occurring Cu(I)–[GSH]₂ complex. Recently, we reported that this complex is able to reduce molecular oxygen into superoxide in a reversible reaction. In the present study, by means of fluorescence, luminescence, EPR and NMR techniques, we investigated the superoxide-generating capacity of the Cu(I)–[GSH]₂ complex, demonstrated the occurrence and characterized the chemical nature of the oxidized complex which is formed upon removing of superoxide radicals from the former reaction, and addressed some of the redox consequences associated with the interaction between the Cu(I)–[GSH]₂ complex, its oxidized complex form, and an in-excess of GSH molecules. The interaction between Cu(I)–[GSH]₂ and added GSH molecules led to an substantial exacerbation of the ability of the former to generate superoxide anions. Removal of superoxide from a solution containing the Cu(I)–[GSH]₂ complex, by addition of Tempol, led to the formation and accumulation of Cu(II)–GSSG. Interaction between the latter complex and GSH molecules permitted the re-generation of the Cu(I)–[GSH]₂ complex and led to a concomitant recovery of its superoxide-generating capacity. Some of the potential redox and biological implications arising from these interactions are discussed.     

In silico and in vitro studies of transition metal complexes derived from curcumin–isoniazid Schiff base

Abstract

A series of transition metal complexes have been synthesized from biologically active curcumin and isoniazid Schiff base. They are characterized by various spectral techniques like UV–Vis, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and mass spectroscopies. Moreover, elemental analysis, magnetic susceptibility and molar conductivity measurements are also carried out. All these data evidence that the metal complexes acquire square planar except zinc(II) which adopts a tetrahedral geometry, and they are non-electrolytic in nature. Groove mode of binding between the calf thymus DNA (CT DNA) and metal complexes is confirmed by electronic absorption titration, viscosity and cyclic voltammetry studies. In addition to that, all the metal complexes are able to cleave pUC 19 DNA. Optimized geometry and ground-state electronic structure calculations of all the synthesized compounds are established out by density functional theory (DFT) using B3LYP method which theoretically reveals that copper(II) complex explores higher stability and higher biological accessibility. This is experimentally corroborated by antimicrobial studies. In silico Absorption, Distribution, Metabolism, Excretion (ADME) studies reveal the biological potential of all synthesized complexes, and also biological activity of the ligand is predicted by PASS online biological activity prediction software. Molecular docking studies are also carried out to confirm the groove mode of binding and receptor–complex interactions.     

Kinetic and equilibrium studies of bile salt–liposome interactions

Abstract

Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC?<?C?≈?TC?<?DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control?<?MKC?<?TC?～?C?<?DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC?<?C?<?DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.     

In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by the impaired functioning of ciliated cells. Its diagnosis is based on the analysis of the structure and functioning of cilia present in the respiratory epithelium (RE) of the patient. Abnormalities of cilia caused by hereditary mutations closely resemble and often overlap with defects induced by the environmental factors. As a result, proper diagnosis of PCD is difficult and may require repeated sampling of patients’ tissue, which is not always possible. The culturing of differentiated cells and tissues derived from the human RE seems to be the best way to diagnose PCD, to study genotype–phenotype relations of genes involved in ciliary dysfunction, as well as other aspects related to the functioning of the RE. In this review, different methods of culturing differentiated cells and tissues derived from the human RE, along with their potential and limitations, are summarized. Several considerations with respect to the factors influencing the process of in vitro differentiation (cell-to-cell interactions, medium composition, cell-support substrate) are also discussed.     

In vitro Antioxidant and Cytotoxic Activities of Extracts of Endemic Tanacetum erzincanense Together with Phenolic Content by LC-ESI-QTOF-MS

In this study, phenolic composition, and in vitro biological activities of ethyl acetate (EAE) and methanol (ME) extracts obtained from the aerial parts of endemic Tanacetum erzincanense were investigated. Total phenolic and flavonoid content of the extracts were determined by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. Antioxidant capacity of the extracts was evaluated over radical scavenging (DPPH and ABTS) and metal ion reducing power (FRAP and CUPRAC) tests. Individual phenolic compounds in ME was analyzed by high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS). Cell inhibitory potential of the extracts was tested against colorectal adenocarcinoma (HT-29), breast adenocarcinoma (MCF-7), and hepatocarcinoma (HepG2) cells by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The results showed that ME contains higher TPC (64.4 mg GAE/g) and TFC (62.2 mg QE/g) than those of EAE (41.5 mg GAE/g and 40.0 mg QE/g). LC-ESI-QTOF/MS analysis revealed that ME is rich in phenolic compounds, namely, chlorogenic acid, apigenin, quercetin, luteolin, and diosmetin. Antioxidant assay results indicated that ME possess stronger activity than EAE and a power that competes with synthetic antioxidants. XTT assay results demonstrated that although both extracts displayed a considerable cytotoxicity against the tested cancer cell lines in a time and dose-dependent manner, ME expressed its selective inhibitory action towards MCF-7 cells with an IC₅₀ value of 20.4 μg/mL for 72 h. These results may serve as a basis for further in vivo studies to examine the potential applications of T. erzincanense in food and pharmaceutical industries.     

In vitro propagation of Eucomis L'Hérit species - plants with medicinal and horticultural potential

Eucomis (Family Hyacinthaceae) are deciduousgeophytes with long, narrow leaves and erect, densely packed flower spikes. Thebulbs are greatly valued in traditional medicine for the treatment of a variety ofailments, and are thus heavily harvested for trade in South Africa's traditional herbal markets. Eucomis species propagaterelatively slowly from offsets and seed, and this, together with theirover-utilisation ethnopharmacologically, has led to their threatened status. Thein vitro propagation of the genusEucomis was investigated to optimise this technique for thebulk production of plants for commercial and conservation purposes. Multipleshoot production was initiated from leaf explants, in all species studied. AMurashige and Skoog (MS) medium, supplemented with 100mgamp;ell;^–1 myo-inositol, 20gamp;ell;^–1 sucrose, and solidified with 2gamp;ell;^–1 Gelrite® was used. Theoptimal hormone combination for shoot initiation in the majority of species was1 mgamp;ell;^–1 NAA and 1mgamp;ell;^–1 BA. Optimal root initiation wasdemonstrated on media supplemented with 1mgamp;ell;^–1 IAA, IBA or NAA, depending onspecies. A continuous culture system using this protocol produced 25–30plantlets per culture bottle, with 10–25 specimens per bottle availablefor acclimatisation. To maximise plantlet survival, different support media usedduring the acclimatisation process were necessary. Certain species respondedbest on a vermiculite medium, while perlite (which holds less water) wasnecessary for the optimal survival rate of other species. Acclimatised plantletswere repotted in a sand: soil mix (1:1).