Β-aminoethyl Ether Research Articles

Light Emission from Fe2+-EGTA-H2O2 System Depends on the pH of the Reaction Milieu within the Range That May Occur in Cells of the Human Body.

A Fe2+-EGTA(ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid)-H2O2 system emits photons, and quenching this chemiluminescence can be used for determination of anti-hydroxyl radical (•OH) activity of various compounds. The generation of •OH and light emission due to oxidative damage to EGTA may depend on the buffer and pH of the reaction milieu. In this study, we evaluated the effect of pH from 6.0 to 7.4 (that may occur in human cells) stabilized with 10 mM phosphate buffer (main intracellular buffer) on a chemiluminescence signal and the ratio of this signal to noise (light emission from medium alone). The highest signal (4698 ± 583 RLU) and signal-to-noise ratio (9.7 ± 1.5) were noted for pH 6.6. Lower and higher pH caused suppression of these variables to 2696 ± 292 RLU, 4.0 ± 0.8 at pH 6.2 and to 3946 ± 558 RLU, 5.0 ± 1.5 at pH 7.4, respectively. The following processes may explain these observations: enhancement and inhibition of •OH production in lower and higher pH; formation of insoluble Fe(OH)3 at neutral and alkaline environments; augmentation of •OH production by phosphates at weakly acidic and neutral environments; and decreased regeneration of Fe2+-EGTA in an acidic environment. Fe2+-EGTA-H2O2 system in 10 mM phosphate buffer pH 6.6 seems optimal for the determination of anti-•OH activity.

Development of turn-on fluorescent sensor based on green emissive carnosine-modified tannic acid carbon dots for the selective detection of ascorbic acid

Ascorbic acid (AA) is an indispensable biological antioxidant with anti-aging potential in neurological disorders such as Parkinson’s and Alzheimer’s. Its abnormal concentrations in physiological fluids like human serum can lead to neurological complications. In this study, a turn-on fluorescent sensor was developed using dipeptide (i.e., carnosine) modification of the polyphenolic tannic acid (TA) carbon dots (CDs) (TATA-CDs) for AA detection. Blue-fluorescent TATA-CDs were synthesized using the hydrothermal treatment of the TA and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) as a nitrogen (N) dopant. TATA-CDs were functionalized with carnosine (TATA-Car CDs) using a facile stirring reaction. Their morphological and optical properties were studied via TEM, XRD, UV–Vis, and fluorescence spectroscopy. TATA-Car CDs displayed green fluorescence with excitation and emission wavelength maxima of 400 and 509 nm, respectively. These novel CDs are responsible for the selective turn-on fluorescence sensing of AA (0 to 15 nM) when mixed with copper (Cu2+) ions and deliver a low detection limit of 1.34 nM. The practicality and feasibility of TATA-Car CDs were investigated in human serum samples. From this perspective, the proposed fluorescent sensor is more suitable for the real-time monitoring of AA in different neurological disorders.

Cynarin, a caffeoylquinic acid derivative in artichoke, inhibits exocytotic glutamate release from rat cortical nerve terminals (synaptosomes)

The purpose of this study was to evaluate the effect of cynarin, a caffeoylquinic acid derivative in artichoke, on glutamate release elicited by 4-aminopyridine (4-AP) in rat cortical nerve terminals (synaptosomes). We observed that cynarin decreased 4-aminopyridine-elicited glutamate release, which was prevented by the removal of external free Ca2+ with ethylene glycol bis (β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or the blockade of P/Q-type calcium channels with ω-agatoxin IVA. Molecular docking also revealed that cynarin formed a hydrogen bond with the P/Q-type Ca2+ channel, indicating a mechanism of action involving Ca2+ influx inhibition. Additionally, the inhibitory effect of cynarin on glutamate release is associated with a change in the available synaptic vesicles, as cynarin decreased 4-AP-elicited FM1-43 release or hypertonic sucrose-evoked glutamate release from synaptosomes. Furthermore, the suppression of protein kinase A (PKA) prevented the effect of cynarin on 4-AP-elicited glutamate release. 4-AP-elicited PKA and synapsin I or synaptosomal-associated protein of 25 kDa (SNAP-25) phosphorylation at PKA-specific residues were also attenuated by cynarin. Our data indicate that cynarin, through the suppression of P/Q-type Ca2+ channels, inhibits PKA activation and attenuates synapsin I and SNAP-25 phosphorylation at PKA-specific residues, thus decreasing synaptic vesicle availability and contributing to glutamate release inhibition in cerebral cortex terminals.

ANALYSIS OF SPERMATOZOA QUALITY USING PERCOLL DENSITY GRADIENT CENTRIFUGATION THROUGH THE ADMINISTRATION OF PHOSPHOLIPID + EGTA

The Percoll density gradient centrifugation (PDGC) method is frequently used in the sexing of spermatozoa. However, this method causes damage to the spermatozoa membranes, resulting in a decreased quality of spermatozoa. We analysed the impacts of phospholipid PC (phosphatidylcholine) and EGTA (ethylene glycol bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid) Ca2+ free buffer on the quality of bovine spermatozoa after the PDGC process, using semen from Friesian Holstein (FH) bulls aged 5 - 8 years. The following variables were observed: spermatozoa motility, spermatozoa viability, spermatozoa membrane integrity, spermatozoa that have not experienced capacitation, spermatozoa that have experienced capacitation and spermatozoa that have undergone acrosomal reaction. The results showed that the administration of phospholipid PC + EGTA Ca2+ free buffer to spermatozoa, followed by the PDGC process, could maintain or further improve the values of all variables. In the PDGC process, phospholipid PC 10% + EGTA Ca2+ free buffer at 1 mM was most suitable.

Novel Extraction Method for Combined Lipid and Metal Speciation From Caenorhabditis elegans With Focus on Iron Redox Status and Lipid Profiling

Parkinson´s disease progression is linked to iron redox status homeostasis via reactive oxygen species (ROS) formation, and lipids are the primary targets of ROS. The determination of iron redox status in vivo is challenging and requires specific extraction methods, which are so far tedious and very time-consuming. We demonstrated a novel, faster, and less laborious extraction method using the chelator ethylene glycol l-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) as a stabilizing agent and synthetic quartz beads for homogenization under an argon atmosphere. Additionally, we combined the metal extraction with a well-established lipid extraction protocol using methyl-tert-butyl ether (MTBE) to avoid the problems of lipid precipitation in frozen samples and to determine lipid profiles and metal species from the same batch. The nonextractable matrix, such as the debris, is removed by centrifugation and digested to determine the total metal content of the sample as well. Lipid profiling using RP-LC–MS demonstrated high accordance of the modified extraction method to the reference method, and the organic solvent does not affect the iron redox status equilibrium. Furthermore, rigorous testing demonstrated the stability of the iron redox status equilibrium during the extraction process, secured by complexation, inert atmosphere, fast preparation, and immediately deep frozen extracts.

Effect of Physiological Concentrations of Vitamin C on the Inhibitation of Hydroxyl Radical Induced Light Emission from Fe2+-EGTA-H2O2 and Fe3+-EGTA-H2O2 Systems In Vitro.

Ascorbic acid (AA) has antioxidant properties. However, in the presence of Fe2+/Fe3+ ions and H2O2, it may behave as a pro-oxidant by accelerating and enhancing the formation of hydroxyl radicals (•OH). Therefore, in this study we evaluated the effect of AA at concentrations of 1 to 200 µmol/L on •OH-induced light emission (at a pH of 7.4 and temperature of 37 °C) from 92.6 µmol/L Fe2+—185.2 µmol/L EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)—2.6 mmol/L H2O2, and 92.6 µmol/L Fe3+—185.2 µmol/L EGTA—2.6 mmol/L H2O2 systems. Dehydroascorbic acid (DHAA) at the same range of concentrations served as the reference compound. Light emission was measured with multitube luminometer (AutoLumat Plus LB 953) for 120 s after automatic injection of H2O2. AA at concentrations of 1 to 50 µmol/L and of 1 to 75 µmol/L completely inhibited light emission from Fe2+-EGTA-H2O2 and Fe3+-EGTA-H2O2, respectively. Concentrations of 100 and 200 µmol/L did not affect chemiluminescence of Fe3+-EGTA-H2O2 but tended to increase light emission from Fe2+-EGTA-H2O2. DHAA at concentrations of 1 to 100 µmol/L had no effect on chemiluminescence of both systems. These results indicate that AA at physiological concentrations exhibits strong antioxidant activity in the presence of chelated iron and H2O2.

The anabolic effect of inorganic polyphosphate on chondrocytes is mediated by calcium signalling.

Inorganic polyphosphates (polyP) are polymers composed of phosphate residues linked by energy-rich phosphoanhydride bonds. As polyP can bind calcium, the hypothesis of this study is that polyP enters chondrocytes and exerts its anabolic effect by calcium influx through calcium channels. PolyP treatment of cartilage tissue formed in 3D culture by bovine chondrocytes showed an increase in proteoglycan accumulation but only when calcium was also present at a concentration of 1.5 mM. This anabolic effect could be prevented by treatment with either ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid or the calcium channel inhibitors gadolinium and nifedipine. Calcium and polyP cotreatment of chondrocytes in monolayer culture resulted in calcium oscillations that were polyP chain length specific and were inhibited by gadolinium and nifedipine. The calcium influx resulted in increased gene expression of sox9, collagen type II, and aggrecan which was prevented by treatment with either calphostin, an inhibitor of protein kinase C, and W7, an inhibitor of calmodulin; suggesting activation of the protein kinase C-calmodulin pathway. Tracing studies using 4',6-diamidino-2-phenylindole, Mitotracker Red, and/or Fura-AM staining showed that polyP was detected in the nucleus, mitochondria, and intracellular vacuoles suggesting that polyP may also enter the cell. PolyP colocalizes with calcium in mitochondria. This study demonstrates that polyP requires the influx of calcium to regulate chondrocyte matrix production, likely via activating calcium signaling. These findings identify the mechanism regulating the anabolic effect of polyP in chondrocytes which will help in its clinical translation into a therapeutic agent for cartilage repair.

Study on β-Casein Depleted Casein Micelles: Micellar Stability, Enzymatic Cross-Linking, and Suitability as Nanocarriers.

β-Casein is an amphiphilic protein and thus considered as multilaterally bound in casein micelles. Its polar molecule part, in particular the phosphoserine residues, can interact electrostatically with colloidal calcium phosphate (CCP) to form nanoclusters and its nonpolar molecule part enhances micellar stability by forming hydrophobic bonds to other caseins. Because cooling weakens hydrophobic interactions, a substantial portion of β-casein can be irreversibly removed from the casein micelle by repeated depletion steps, including cooling and subsequent ultracentrifugation. Although this effect of cooling on the micellar β-casein concentration has been well known for decades, the influence of depletion on the main characteristics of casein micelles has been less investigated yet. Therefore, we aimed to analyze the consequences of β-casein depletion on the stability as well as the functionality of casein micelles to evaluate the suitability of depleted compared to native casein micelles as nanocarriers. Up to 43.2% of the total β-casein was irreversibly sequestered from native casein micelles by repeated cooling and ultracentrifugation steps. Depletion showed no effect on size distribution as well as polydispersity and particle concentration of micelle suspensions as measured via dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), respectively. Furthermore, the stability of the micelles against ethanol or the chelating agent ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was not influenced by β-casein depletion. Notwithstanding, depleted micelles were less susceptible to enzymatic cross-linking by microbial transglutaminase (mTG), indicating narrowed water channels due to depletion. Additionally, loading experiments showed that depleted micelles could be loaded with linoleic acid (LA) as intensively as native micelles, whereupon LA displaces up to 81.3% of β-casein from native micelles. Our results confirm that depletion does not enhance the ability of the casein micelle to act as a nanocarrier for hydrophobic substances but could support the understanding of the casein micelle structure. Based on the observed unchanged stability against EGTA, the hindered enzymatical cross-linking, and the efficient displacing of β-casein by LA, we suggest that the major portion of micellar β-casein is hydrophobically incorporated into the micelle structure without impact on the formation of calcium phosphate nanoclusters. The main role of β-casein for the casein micelle structure, therefore, might be to facilitate the high hydration of the interior and thus the high permeability of casein micelles.

Hydrogen Sulfide Relaxes Human Uterine Artery via Activating Smooth Muscle BKCa Channels.

Opening of large conductance calcium-activated and voltage-dependent potassium (BKCa) channels hyperpolarizes plasma membranes of smooth muscle (SM) to cause vasodilation, underling a key mechanism for mediating uterine artery (UA) dilation in pregnancy. Hydrogen sulfide (H2S) has been recently identified as a new UA vasodilator, yet the mechanism underlying H2S-induced UA dilation is unknown. Here, we tested whether H2S activated BKCa channels in human UA smooth muscle cells (hUASMC) to mediate UA relaxation. Multiple BKCa subunits were found in human UA in vitro and hUASMC in vitro, and high β1 and γ1 proteins were localized in SM cells in human UA. Baseline outward currents, recorded by whole-cell and single-channel patch clamps, were significantly inhibited by specific BKCa blockers iberiotoxin (IBTX) or tetraethylammonium, showing specific BKCa activity in hUASMC. H2S dose (NaHS, 1–1000 µM)-dependently potentiated BKCa currents and open probability. Co-incubation with a Ca2+ blocker nifedipine (5 µM) or a chelator (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 5 mM) did not alter H2S-potentiated BKCa currents and open probability. NaHS also dose-dependently relaxed phenylephrine pre-constricted freshly prepared human UA rings, which was inhibited by IBTX. Thus, H2S stimulated human UA relaxation at least partially via activating SM BKCa channels independent of extracellular Ca2+.

Substrate curvature induces fallopian tube epithelial cell invasion via cell-cell tension in a model of ovarian cortical inclusion cysts.

Throughout the body, epithelial tissues contain curved features (e.g. cysts, ducts and crypts) that influence cell behaviors. These structures have varied curvature, with flat structures having zero curvature and structures such as crypts having large curvature. In the ovary, cortical inclusion cysts (CICs) of varying curvatures are found, and fallopian tube epithelial (FTE) cells have been found trapped within these cysts. FTE are the precursor for ovarian cancer, and the CIC niche has been proposed to play a role in ovarian cancer progression. We hypothesized that variations in ovarian CIC curvature that occur during cyst resolution impact the ability of trapped FTE cells to invade into the surrounding stroma. Using a lumen model in collagen gels, we determined that increased curvature resulted in more invasions of mouse FTE cells. To isolate curvature as a system parameter, we developed a novel technique to pattern concave curvatures into collagen gels. When FTE cells were seeded to confluency on curved substrates, increases in curvature increased the number of invading FTE cells and the invasion distance. FTE invasion into collagen substrates with higher curvature depended on matrix metalloproteinases (MMPs), but expression of collagen I degrading Mmps was not different on curved and flat regions. A finite-element model predicted that contractility and cell-cell connections were essential for increased invasion on substrates with higher curvature, while cell-substrate interactions had minimal effect. Experiments supported these predictions, with invasion decreased by blebbistatin, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or N-cadherin-blocking antibody, but with no effect from a focal adhesion kinase inhibitor. Finally, experimental evidence supports that cell invasion on curved substrates occurs in two phases-a cell-cell-dependent initiation phase where individual cells break away from the monolayer and an MMP-dependent phase as cells migrate further into the collagen matrix.

Effect of HEDTA, CDTA, and EGTA Chelates on Remediation of Pb- and Zn-Polluted Soil Using Sunflower and Canola Plants: A Case Study; Soil Around Bama Pb and Zn Mine

Background: The soil heavy metal pollution caused by human activities is one of the main environmental problems. Therefore, it is necessary to use appropriate methods to remediate such soils. Objectives: The current study aimed at investigating the effect of Hydroxyl Ethylene Diamine Tetra Acetic Acid (HEDTA), Trans-1,2-Cyclohexylene Dinitrilo Tetra Acetic Acid (CDTA), and Ethylene Glycol-bis (β-Aminoethyl Ether)-N,N,N’,N’-Tetra Acetic Acid (EGTA) chelates on Pb and Zn removal efficiency in a Pb and Zn contaminated soil. Methods: This research was performed as a factorial experiment in a randomized complete block design with four levels of chelate application rate (0, 1.5, 3, and 4.5 mM/kg soil), two levels of chelates application times (one or two weeks before harvesting), and plant types (sunflower or canola). At the end of the experiment, Pb and Zn concentrations in the soil and plant samples were measured and the least significant difference (LSD) test was employed to determine the differences between the means. Results: The effectiveness of studied chelates on the availability of Zn and Pb in soil was in the descending order as HEDTA > CDTA > EGTA. Application of 4.5 mM/kg soil of CDTA chelate under sunflower and canola cultivation caused a significant increase in the availability of Zn and Pb in soil compared to 1.5 mM/kg by 12.2% and 13.3%, respectively. Plant type also had a significant effect on increasing the availability of Pb and Zn in soil. Conclusions: Plant type, application rate, and chelates type are important factors on remediation of heavy metals in contaminated soil. However, the role of physicochemical properties of soil on its heavy metal availability cannot be ignored.

Characterization of a novel thermostable carboxylesterase from thermoalkaliphilic bacterium Bacillus thermocloaceae.

A novel thermostable carboxylesterase (Est5250) of thermoalkaliphilic bacterium Bacillus thermocloaceae was heterologously expressed in Escherichia coli and its biochemical properties were investigated. Est5250 showed optimum esterase activity at 60 °C and pH 8.0. The enzyme was highly thermostable at 60 °C, interestingly, the thermostability was enhanced in the presence of Ca2+, retaining more than 60% of its original activity after 12h of pre-incubation. Est5250 was active in the presence of 1% (v/v) of organic solvents and 0.1% (v/v) of non-ionic detergents. The enzyme activity was significantly enhanced up to 167% and 159% in the presence of 2-mercaptoethanol and dithiothreitol, respectively. Est5250 showed high substrate specificity for short-chain p-nitrophenyl-esters. Kinetic constants, Km and kcat, for p-nitrophenyl-acetate were 185.8μM and 186.6s-1, respectively. Est5250 showed outstanding thermostability and tolerance to various organic solvents under thermoalkaliphilic conditions, suggesting that it would be a highly suitable biocatalyst for various biotechnological applications. Abbreviations: B. thermocloaceae sp.: Bacillus thermocloaceae; E. coli: Escherichia coli; NP: nitrophenyl; DMSO: dimethyl sulfoxide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMF: dimethyl formamide; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; CTAB: cetrimonium bromide; PMSF: phenylmethylsulfonyl fluoride; DEPC: diethyl pyrocarbonate; 2-ME: 2-mercaptoethanol; DTT: dithiothreitol.

Reveal the Role of Store‐Operated Calcium Entry in Epirubicin‐induced Acute Oxidative Stress in Cardiomyocytes

Epirubicin (EPI) is a widely used anthracycline chemotherapy drug, whose use is limited by its side effects including cardiomyopathy, hypertrophy and chronic heart failure. The cardiotoxicity of EPI has been linked to the generation of reactive oxygen species (ROS) in mitochondria and disruption of cardiac ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum membrane. As Store‐Operated Calcium Entry (SOCE) is important in regulating intracellular Ca2+ signaling, we tested whether dysregulated SOCE was also involved in EPI‐induced acute cardiotoxicity. This study compared intracellular ROS levels using fluorescent probe dihydroethidium (DHE) in EPI‐treated HL‐1, an adult mouse atrial cardiomyocyte cell line. Treatment of EPI at 1uM for 30min significantly increased intracellular ROS levels in HL‐1 compared with vehicle‐treated cells (3.8fold, p<0.0001). HL‐1 cells were then treated with 20uM BTP2, a potent SOCE inhibitor, for 30min. However, BTP2 failed to significantly decrease the EPI‐induced intracellular ROS (p=0.9987, n=8 replicates). In addition, 50uM ethylene glycol‐bis (β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (EGTA‐AM) failed to prevent EPI‐induced ROS increase (p=0.9768, n=8 replicates). These results might be limited by the technical challenge such as DHE not able to quantitatively distinguish intracellular ROS levels and EGTA‐AM might not be fast enough to chelate Ca2+ from Ca2+ influx. Ongoing project will seek to overcome the technical limitation to further test the role of SOCE in EPI‐induced acute cardiotoxicity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Light emission from the Fe2+–EDTA–ascorbic acid–H2O2 system strongly enhanced by plant phenolic acids

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra-weak photon emission (UPE). We investigated the UPE from the Fe2+ -EDTA (ethylenediaminetetraacetic acid)-AA (ascorbic acid)-H2 O2 (hydrogen peroxide) system with a multitube luminometer (Peltier-cooled photon counter, spectral range 380-630nm). The UPE, of 92.6μmol/L Fe2+ , 185.2μmol/L EDTA, 472μmol/L AA, 2.6mmol/L H2 O2 , reached 1217±118 relative light units during 2min measurement and was about two times higher (P<0.001) than the UPE of incomplete systems (Fe2+ -AA-H2 O2 , Fe2+ -EDTA-H2 O2 , AA-H2 O2 ) and medium alone. Substitution of Fe2+ with Cr2+ , Co2+ , Mn2+ or Cu2+ as well as of EDTA with EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or citrate powerfully inhibited UPE. Experiments with scavengers of reactive oxygen species (dimethyl sulfoxide, mannitol, sodium azide, superoxide dismutase) revealed the dependence of UPE only on hydroxyl radicals. Dimethyl sulfoxide at the concentration of 0.74mmol/L inhibited UPE by 79±4%. Plant phenolics (ferulic, chlorogenic and caffec acids) at the concentration of 870μmol/L strongly enhanced UPE by 5-, 13.9- and 46.8-times (P<0.001), respectively. It is suggested that augmentation of UPE from Fe2+ -EDTA-AA-H2 O2 system can be applied for detection of these phytochemicals.

Monitoring glucose, calcium, and magnesium levels in saliva as a non-invasive analysis by sequential injection multi-parametric determination

The use of saliva for diagnose and surveillance of systemic illnesses, and general health has been arousing great interest worldwide, emerging as a highly desirable goal in healthcare. The collection is non-invasive, stress-free, inexpensive, and simple representing a major asset. Glucose, calcium, and magnesium concentration are three major parameters evaluated in clinical context due to their essential role in a wide range of biochemical reactions, and consequently many health disorders. In this work, a spectrophotometric sequential injection method is described for the fast screening of glucose, calcium, and magnesium in saliva samples. The glucose determination reaction involves the oxidation of the aldehyde functional group present in glucose with simultaneous reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino, 5-nitrosalicylic acid under alkaline conditions, followed by the development of colour. The determination of both metals is based on their reaction with cresolphtalein complexone (CPC), and the interference of calcium in the magnesium determination minimized by ethylene glycol-bis[β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid (EGTA). The developed multi-parametric method enabled dynamic ranges of 50 – 300 mg/dL for glucose, 0.1 − 2 mg/dL for calcium, and 0.1 − 0.5 mg/dL for magnesium. Determination rates of 28, 60, 52 h−1 were achieved for glucose, calcium, and magnesium, respectively. Less than 300 µL of saliva is required for the multi-parametric determination due to saliva viscosity and inherent necessity of dilution prior to analysis. RSDs lower than 5% were obtained, and the results agreed with those obtained by reference methods, while recovery tests confirmed its accuracy.

Light Emission from the Fe2+-EGTA-H₂O₂ System: Possible Application for the Determination of Antioxidant Activity of Plant Phenolics.

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra-weak photon emission (UPE). We investigated the UPE from the Fe2+-EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)–H2O2 system with a multitube luminometer (Peltier-cooled photon counter, spectral range 380 to 630 nm). The UPE of 92.6 µmol/L Fe2+—185.2 µmol/L EGTA—2.6 mmol/L H2O2 reached 4319 ± 755 relative light units during 2 min measurement and was about seven times higher (p < 0.001) than the UPE of incomplete systems (Fe2+-H2O2, EGTA-H2O2) and medium alone. Substitution of Fe2+ with Cr2+, Co2+, Mn2+ or Cu2+ as well as of EGTA with EDTA (ethylenediaminetetraacetic acid) or citrate completely abolished UPE. Experiments with ROS scavengers revealed the dependence of UPE on hydroxyl radicals suggesting occurrence of oxidative attack and cleavage of the ether bond in EGTA backbone structure and formation of triplet excited carbonyl groups with subsequent light emission. Plant phenolics (ferulic, chlorogenic and caffec acids) at concentration 87 µmol/L and ascorbate at 0.46 mmol/L inhibited UPE by 90 ± 4%, 90 ± 5%, 97 ± 2% and 92 ± 1%, respectively. Quenching of UPE from Fe2+-EGTA-H2O2 system can be used for evaluation of antioxidant activity of phytochemicals.

Core-Shell Silver Nanoparticles in Endodontic Disinfection Solutions Enable Long-Term Antimicrobial Effect on Oral Biofilms.

To achieve effective long-term disinfection of the root canals, we synthesized core-shell silver nanoparticles (AgNPs@SiO2) and used them to develop two irrigation solutions containing sodium phytate (SP) and ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), respectively. Ex vivo studies with instrumented root canals revealed that the developed irrigation solutions can effectively remove the smear layer from the dentinal surfaces. Further in vitro experiments with single- and multispecies biofilms demonstrated for the first time that AgNPs@SiO2-based irrigation solutions possess excellent antimicrobial activities for at least 7 days, whereas the bare AgNPs lose the activity almost immediately and do not show any antibacterial activity after 2 days. The long-term antimicrobial activity exhibited by AgNPs@SiO2 solutions can be attributed to the sustainable availability of soluble silver, even after 7 days. Both solutions showed lower cytotoxicity toward human gingival fibroblasts compared to the conventionally used solution (3% NaOCl and 17% EDTA). Irrigation solutions containing AgNP@SiO2 may therefore be highly promising for applications needing a long-term antimicrobial effect.

Two-step chromatographic purification of glutathione S-transferase-tagged human papillomavirus type 16 E6 protein and its application for serology

Human papillomavirus (HPV) E6 protein is an oncoprotein with a pivotal role in cervical carcinogenesis. Expression and purification of HPV E6 from Escherichia coli (E. coli) has been difficult because of its strong hydrophobicity even when expressed as a fusion protein with glutathione S-transferase (GST). There has been no protocol suggested for purifying GST-tagged HPV E6 protein with high purity so far. Herein, we provide efficient protocol for purifying GST-HPV16 E6 protein for the first time. In the current study, the GST-tagged protein was expressed in E. coli and a purification method was designed using cation-exchange chromatography followed by GST-affinity chromatography. Using physiological pH buffer during cell lysis and first cation-exchange chromatography significantly reduced yield of full-length GST-HPV16 E6 protein. It was found that using an alkaline buffer during cation-exchange chromatography was needed to obtain full length GST-HPV16 E6 protein. GST-HPV16 E6 protein recovered from the purification using alkaline condition retained its inherent p53-binding ability. Moreover, we were able to detect anti-HPV16 E6 antibodies with high sensitivity in sera from patients with cervical cancer using the GST–HPV16 E6 protein. It was found that the GST-HPV16 E6 protein could be used as a coating agent to enhance the sensitivity of detection of serum anti-HPV16 E6 antibodies when treated with ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N'-tetraacetic acid (EGTA). These results indicate that the two-step chromatographic purification allows obtaining high purity of GST-HPV16 E6 protein and the GST-HPV16 E6 is suitable to be used as an antigen of serology assay.

Ca2+ involvement in activation of extracellular-signal-regulated-kinase 1/2 and m-calpain after axotomy of the sciatic nerve.

Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells (SCs), are poorly understood. Here, we investigated axotomy-induced activation of extracellular-signal-regulated kinase-1/2 (ERK1/2; important for proliferation) and m-calpain in vitro, and the relation to Ca2+ deletion and Schwann cell proliferation and death after rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis(β-aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA). In some experiments, 5-bromo-2’-deoxyuridine (BrdU) was added during the last 24 hours to detect proliferating cells and propidium iodide (PI) was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2. The experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A significant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca2+, activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca2+ deprivation also significantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca2+ seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca2+ levels are also required for m-calpain activation and up-regulation in axons. Thus, regulation of Ca2+ levels is likely to be a useful method to promote SC proliferation.

Effects of Extracellular Calcium and 1,25 dihydroxyvitamin D3 on Sebaceous Gland Cells In vitro and In vivo

Calcium and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) are promoters of epithelial cell functions; however their effects on sebaceous glands are unknown. In this study, morphology, ultrastructure, cell numbers, lipid synthesis and apoptosis of SZ95 sebocytes were assessed in vitro under different concentrations of extracellular calcium with or without 1,25(OH)2D3. Moreover, serum calcium and 1,25(OH)2D3 levels were assessed in acne and non-acne patients (controls). Under conditions of low extracellular calcium, lipogenesis and cell detachment were observed. Increasing extracellular calcium enhanced sebocyte numbers, induced epithelial morphology and reduced lipogenesis. Moreover, a reduction in extracellular calcium reduced E-cadherin and enhanced caspase 3/7 activity (apoptosis), whereas calcium chelation by EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) resulted in enhanced lipogenesis. 1,25(OH)2D3 decreased sebaceous lipogenesis, but also induced signs of autophagy. In the clinical study, patients and controls exhibited normal serum calcium levels. Younger acne patients presented lower 1,25(OH)2D3 levels than did older ones. In conclusion, extracellular calcium and 1,25(OH)2D3 regulate sebocyte morphology, increase cell numbers, decrease sebaceous lipogenesis and induce cell autophagy in vitro. The increased ionized calcium and the reduced 1,25(OH)2D3 levels detected in the serum of younger patients with acne may contribute respectively to increased sebaceous gland volume and enhanced lipogenesis.