E Prostanoid-1 receptor regulates renal medullary αENaC in rats infused with angiotensin II

E Prostanoid (EP) receptors play an important role in urinary Na⁺ excretion. In the kidney, the epithelial sodium channel (ENaC) is the rate-limiting-step for Na⁺ reabsorption. We hypothesized that activation of EP1/EP3 regulates the expression of ENaC in the face of renin-angiotensin-aldosterone-system (RAAS) activation. In primary cultures of inner medullary collecting duct (IMCD) cells, sulprostone (EP1 > EP3 agonist, 1 μM) and 17 Phenyl trinor (17 Pt, EP1 agonist, 10 μM) prevented the up-regulation of αENaC mRNA induced by aldosterone (10 nM). In Sprague-Dawley rats infused with angiotensin II (0.4 μg/kg/min), αENaC expression was up-regulated in renal cortex and medulla coincidently with high plasma aldosterone levels. Sulprostone and/or 17 Pt prevented this effect in renal medulla but not in cortex. Immunocytochemistry demonstrated that IMCD cells express EP1. Our results suggest that specific activation of EP1 receptor during RAAS activation antagonizes the action of aldosterone on αENaC expression in the renal medulla.     

In vitro effects of bisphenol A on the quality parameters,oxidative stress,DNA integrity and adenosine triphosphate content in sterlet (Acipenser ruthenus) spermatozoa

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be responsible for adverse effect on reproductive ability of various species. In the present study the effect of BPA on the quality parameters, oxidative stress, the DNA integrity and intracellular ATP content of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were exposed to concentrations of BPA possibly occurring in nature (0.5, 1.75, 2.5, 5 and 10 μg/L) for 2 h. Results revealed that BPA significantly decreased spermatozoa motility and velocity of spermatozoa at concentration of BPA 2.5–10 μg/L. Significant positive correlation (r = 0.713, P < 0.05) was found between percent motile spermatozoa and ATP content. Oxidative stress was observed at concentrations 1.75–10 μg/L, as reflected by significantly higher levels of protein and lipid oxidation and superoxide dismutase activity. Intracellular ATP content of spermatozoa decreased with increasing concentrations of BPA. A dramatic increase in DNA fragmentation expressed as percent tail DNA (2.2% ± 0.46) and Olive tail moment (0.37 ± 0.09 arbitrary units) was recorded at concentrations of 1.75 μg/L and above. The present study confirms that concentrations of BPA that can be encountered in nature are capable to induce oxidative stress, leading to impaired sperm quality, DNA fragmentation and intracellular ATP content.     

Inhibitory effect of glycoprotein isolated from Opuntia ficus-indica var. saboten MAKINO on activities of allergy-mediators in compound 48/80-stimulated mast cells

The present study was performed to investigate the anti-allergy potentials of glycoprotein (90 kDa) isolated from Opuntia ficus-indica var. saboten MAKINO (OFI glycoprotein) in vivo (ICR mice) and in vitro (RBL-2H3 cells). At first, to know whether the OFI glycoprotein has an inhibitory ability for allergy in vivo, we evaluated the activities of allergy-related factors such as histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in compound 48/80 (8 ml/kg BW)-treated ICR mice. After that, we studied to found the effect for anti-allergy in vitro such as nuclear factor kappa B (NF-κB) and inducible nitric oxide synthase (iNOS), extracellular signal-regulated kinase (ERK) 1/2, arachidonic acid, and cyclooxygenase-2 (COX-2) in compound 48/80 (5 μg/ml)-treated RBL-2H3 cells. Our results showed that the OFI glycoprotein (5 mg/kg) inhibited histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in mice serum. Also OFI glycoprotein (25 μg/ml) has suppressive effects on the expression of MAPK (ERK1/2), and on protein expression of anti-allergic proteins (iNOS and COX-2). Thus, we speculate that the OFI glycoprotein is an example of natural compound that blocks anti-allergic signal transduction pathways.     

Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells

Background

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.

Methods

Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.

Results

10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD^(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.

Conclusions

1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.

General significance

Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.     

In vitro assessment of plant lectins with anti-pinwood nematode activity

Two lectin proteins were purified from the corms of Pinellia ternata and Lycoris radiata. Both P. ternata agglutinin (PTA) protein and L. radiata agglutinin (LRA) protein formed polymers and coagulated both rabbit red blood cells and yeast cells. The two proteins were each diluted to different concentration and then mixed with pinewood nematodes, and nematode survival was measured. Results showed that the two lectin proteins showed significant levels of resistance against nematodes and the nematode population was significantly reduced, compared to PBS buffer without protein control group. The mean number of nematodes of two lectin proteins group was significantly lower than that of control group constantly throughout the assay period with differences being very significant at P < 0.01 after 24 h. After 96 h, when 500 μg/ml proteins were used, nematode number significantly declined to an average of 26 (approximately 43% of the controls) and 32.2 (approximately 53.3% of the controls) nematodes at LRA and PTA protein, respectively, compared to the control group. Results also indicated that higher concentrations of protein were more toxic to the pinewood nematode. Even when the concentration was as low as 30 μg/ml, the toxic proteins retained their anti-nematode activity. Furthermore, pinewood nematode was exposed to the proteins for longer, more pinewood nematodes were killed. Our results indicated the two lectin proteins both apparently have a toxic effect on the pinewood nematode that affects its survival in vitro.     

Quantification of folate in fruits and vegetables: A fluorescence-based homogeneous assay

A high-throughput, homogeneous, fluorescence polarization, and fluorescence intensity assay has been developed for the measurement of folate in fruits and vegetables. This assay is based on the competitive displacement of the fluorescent folate ligands Alexa Fluor (Alexa) 594-folate and Alexa 660-folate from bovine milk folate-binding protein by folates in fruit and vegetable extracts. These fluorescent ligands are employed because their excitation and emission maxima are in regions of the spectrum with minimal autofluorescence in many extracts. Folate-binding protein and Alexa-folate were typically used at concentrations of 0.5 μg/ml and 5 nM, respectively, in 20-μl volumes in 384-well microplates. The assay is complete within 100 min. The folate estimate is unaffected by the heterogeneity of polyglutamyl residues that complicates the liquid chromatography-mass spectrometry (LC-MS)-based methods of quantification. In this assay, folic acid had an apparent affinity 2.5-fold greater than 5-methyltetrahydrofolate (5MTHF); therefore, it cannot be used to quantify folate when both natural and synthetic folate are present. 5MTHF-equivalent values were measured in broccoli (240 μg/100 g), strawberry (113 μg/100 g), white grape (32 μg/100 g), orange (44 μg/100 g), tomato (12 μg/100 g), raspberry (31 μg/100 g), banana (29 μg/g), and kiwifruit (36 μg/100 g). These data are similar to published values. However, the assay will not detect 5-formyltetrahydrofolate which is a significant constituent of the total folate in lettuce, spinach, carrot, and peppers.     

A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin

A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 μM (26.1 μg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 μM (∼0.18 μg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 μL in 384-well microplates.     

Preparation of high-capacity supports containing protein G immobilized to porous silica

This study examined the preparation of high-capacity silica supports containing immobilized protein G. A maximum content of 39 mg protein G/g silica was obtained when using 100 Å pore size silica, followed by 33 mg/g for 50 Å silica and 9.3-24 mg/g for 300-4000 Å silica. The surface coverage of protein G increased with pore size, with a maximum level of 0.037 μmol/m² being obtained for 4000 Å silica. These supports gave comparable apparent activities (i.e., 30-47% binding to rabbit immunoglobulin G [IgG]), with the highest binding capacities (71-77 mg IgG/g silica) being obtained for 50-100 Å silica.     

Advanced glycation end-product-inhibited cell proliferation and protein expression of beta-catenin and cyclin D1 are dependent on glycogen synthase kinase 3beta in LLC-PK1 cells

Glycogen synthase kinase 3β (GSK3β) is increased by high glucose in mesangial cells. Thus, we studied the role of GSK3β in advanced glycation end-product (AGE)-induced effects in the proximal tubule-like LLC-PK1 cells. We found that AGE (100 μg/ml) time-dependently (8-48 h) increased phospho-GSK3β-Tyr216 (active GSK3β) and time-dependently (4-24 h) decreased phospho-GSK3β-Ser21/9 (inactive GSK3β) protein expression. Meanwhile, AGE (100 μg/ml) activated GSK3β kinase at 8-48 h. AGE (100 μg/ml) dose-dependently (75-100 μg/ml) decreased β-catenin protein expression but AGE did not decrease β-catenin protein expression until 48 h. SB216763 (a GSK3β inhibitor) and GSK3β shRNA attenuated AGE (100 μg/ml)-inhibited cell proliferation and protein expression of β-catenin and cyclin D1 at 48 h. SB216763 also attenuated AGE-induced type IV collagen. We conclude that AGE activates GSK3β in LLC-PK1 cells. AGE-inhibited β-catenin and cyclin D1 protein expression are dependent on GSK3β. Moreover, AGE-inhibited cell proliferation and AGE-induced type IV collagen protein expression are dependent on GSK3β.     

Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1-10 μM). The protein kinase A inhibitor KT5720 (1 μM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction.     

Determination of nanograms of proteins based on decreased resonance light scattering of zwitterionic gemini surfactant

A new high-sensitivity detection of protein assay at the nanogram level is proposed based on the decreased resonance light scattering (RLS) signals of zwitterionic gemini surfactant (phosphodiesters quaternary ammonium salt [PQAS]). It was found that PQAS self-assembled into nanometer-scale PQAS aggregates, which induced intense RLS signal in Britton-Robinson (BR) buffer solution (pH 10.5). Under the optimum conditions, the RLS intensity quenching extent of PQAS aggregation was in proportion to the concentration of proteins in the range of 0.0012-1.08 μg/ml for bovine serum albumin, 0.0015-0.95 μg/ml for human serum albumin, and 0.0025-1.3 μg/ml for γ-globulin. The detection limits were 0.8, 1.2, and 2.0 ng/ml, respectively. The proposed method was successfully applied to determine total protein in human serum samples, and the results were identical to those obtained by the Bradford assay. The mechanism of interaction between PQAS and protein was studied using RLS, fluorescence, and time-resolved fluorescence, which indicated that the new complex formed between them, disaggregating self-aggregation of PQAS, resulted in the dominated quenching of RLS signal of the assay system.     

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 ± 28 μg/l) compared with control healthy subjects (mean 131 ± 10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.     

Simultaneous determination of isoniazid,rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.     

Quantitative enzyme-linked immunosorbent assay determination of an abundant hemoglobin-derived anti-infective peptide in human placenta

A fragment of the human β-chain of hemoglobin (HEM), hHEMβ111-146, was shown to have broad antimicrobial properties. The 3.9-kDa peptide was postulated to occur in high concentrations in placenta tissue. We established a reliable method to quantify hHEMβ111-146 in placenta tissue. Our methodology consists of a tissue extraction step (step 1), a chromatographic enrichment step (step 2), and a final quantification step (step 3) by enzyme-linked immunosorbent assay (ELISA). The specificity of the ELISA reaction was confirmed by parallel analysis of the samples via Western blot (step 4). The ELISA measured the absorbance of a tetramethylbenzidine substrate at 450 nm. It showed no cross-reactivity with the corresponding γ- and α-HEM regions and low cross-reactivity with the β-HEM region and full-length HEM. The sample preparation procedure enabled a prepurification of hHEMβ111-146, completely eliminating cross-reactive proteins and HEM peptides. The linear range of detection in step 3 was 20-200 ng/well (200-2000 μg/L) with a limit of quantification of 23 ng/well (230 μg/L) and a limit of detection of 7 ng/well (70 μg/L). The assay was characterized by good linearity (r² > 0.99), intraday precision (coefficient of variation [CV] = 2.2-8.3%), interday precision (CV = 1.8-9.1%), and accuracy (76-109%). The mean recovery of the ELISA was determined to be 97%, and the overall recovery during steps 1-3 was found to be 40.3 ± 2.5%. We measured concentrations from 0.28 to 0.74 mg/g placenta tissue of the hHEMβ111-146 in different placenta samples with an average concentration of 0.57 mg/g. This abundant concentration supports an important physiological role of hHEMβ111-146 in the placenta infective barrier.     

Inhibition of mammalian thioredoxin reductase by black tea and its constituents: Implications for anticancer actions

Black tea is recently reported to have anti-carcinogenic effects through pro-oxidant property, but the underlying mechanisms remain unclear. Mammalian cytosolic thioredoxin reductase (TrxR1) is well -known for its anti-oxidation activity. In this study, we found that black tea extract (BTE) and theaflavins (TFs), the major black tea polyphenols, inhibited the purified TrxR1 with IC₅₀ 44 μg/ml and 21 ± 1 μg/ml, respectively. Kinetics of TFs exhibited a mixed type of competitive and non-competitive inhibition, with K_is 4 ± 1 μg/ml and K_ii 26 ± 5 μg/ml against coenzyme NADPH, and with K_is 12 ± 3 μg/ml and K_ii 27 ± 5 μg/ml against substrate DTNB. In addition, TFs inhibited TrxR1 in a time-dependent manner. In an equilibrium step, a reversible TrxR1-TFs complex (E * I) forms, which is followed by a slow irreversible first-order inactivation step. Rate constant of the inactivation was 0.7 min⁻¹, and dissociation constant of E * I was 51.9 μg/ml. Treatment of NADPH-reduced TrxR1 with TFs decreased 5-(Iodoacetamido) fluorescein incorporation, a fluorescent thiol-reactive reagent, suggesting that Sec/Cys residue(s) in the active site may be involved in the binding of TFs. The inhibitory capacity of TFs depends on their structure. Among the TFs tested, gallated forms had strong inhibitory effects. The interactions between TFs and TrxR1 were investigated by molecular docking, which revealed important features of the binding mechanism of theaflavins. An inhibitory effect of BTE on viability of HeLa cells was observed with IC₅₀ 29 μg/ml. At 33 μg/ml of BTE, TrxR1 activity in HeLa cells was decreased by 73% at 22 h after BTE treatment. TFs inhibited cell viability with IC₅₀ 10 ± 4 μg/ml for HeLa cells and with IC₅₀ 20 ± 5 μg/ml for EAhy926 cells. The cell susceptibility to TFs was inversely correlated to cellular levels of TrxR1. The inhibitory actions of TFs on TrxR1 may be an important mechanism of their anti-cancer properties.     

Key genes and proteins involved in CTCM-reducing microvascular endothelial cell permeability induced by SLT-IIv using gene chips and DIGE

An Affymetrix mouse genome array and differential in-gel electrophoresis (DIGE) techniques were used to investigate the pharmacological mechanisms of a mixture of herbs, designated CTCM, a compound of traditional Chinese medicine, for the treatment of increased permeability in mouse intestinal microvascular endothelial cells (MIMECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMECs were challenged with 10 μg/ml SLT-IIv for 12 h and then treated with CTCM at a concentration of 200 μg/ml for 12 h. Total RNA and proteins from each treatment group were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip® Mouse Genome 430 2.0 microarray and DIGE. The results obtained demonstrated that there were one genes downregulated and one genes upregulated, one protein downregulated and four proteins upregulated in the SLT-IIv group compared to the control group. In the CTCM group, four genes were upregulated, three genes were downregulated, a single protein was downregulated and a single protein was upregulated when compared to the control group. When the CTCM-treated group was compared to the SLT-IIv group, expression of one gene was found to be increased, and all other genes were decreased, with five proteins downregulated. Analysis of the data suggested that CTCM specifically and effectively reduced microvascular endothelial cell permeability to SLT-IIv in the treatment of pig edema disease. In the CTCM-treated group, hspa9 expression was increased in both gene chip and DIGE analysis, so it may be a key protein in reducing cell permeability and utilized in medical treatments.     

Sensitive detection of C-reactive protein in serum by immunoprecipitation–microchip capillary gel electrophoresis

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP–MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R² = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP–MCGE bears potential for point-of-care diagnosis.     

Recovery of fat snook, Centropomus parallelus (Teleostei: Perciformes) after subchronic exposure to copper

We studied the recovery of juvenile fat snook (Centropomus parallelus) after subchronic exposure to different concentrations of copper. Healthy juveniles (1.98 g) were exposed to 25 or 50 μg Cu/L for 30 days (12 replicates with 5 fish in each one), and recovery was observed at 0, 4, 10, and 30 days after exposure (3 replicates with 5 fish in each one). Copper genotoxicity in exposed individuals was observed using a micronucleus assay, and recovery was not observed even 30 days post-exposure. Copper accumulation was observed in fish exposed to 25 or 50 μg/L of copper in the gills (14.4 and 34.4 μg/g, respectively) and muscle (5.7 and 5.5 μg/g, respectively), and a return to normal copper levels (6.0 μg/g for gills and 2.5 μg/g for muscle) was observed 4 and 30 days post-exposure in the gills and muscle tissues, respectively. Glutathione S-transferase (GST) was 80% inhibited in individuals exposed to copper and returned to normal levels for fish exposed to basal concentrations within 10 days. Although copper accumulation in tissues dispersed 30 days post-exposure, no recovery from genotoxicity was observed during this time. Thirty days was not enough to recover juvenile fat snook following subchronic exposure to copper.     

Development of a high-throughput method for the determination of ethosuximide in human plasma by liquid chromatography mass spectrometry

A simple, rapid, sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantification of ethosuximide in human plasma is described. Analyte was chromatographed on a Hypersil Gold C₁₈ column (100 mm × 2.1 mm, i.d., 1.9 μm) with isocratic elution at a flow rate of 0.250 mL/min and pravastatin was used as the internal standard. The assay involves a simple solid-phase extraction procedure of 0.25 mL human plasma and the analysis was performed on a triple-quadrupole tandem mass spectrometer by MRM mode via electrospray ionization (ESI). The method was linear in the concentration range of 0.25–60.0 μg/mL. The lower limit of quantification (LLOQ) was 0.25 μg/mL. The within- and between-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 95.1% and 94.4% for ethosuximide and pravastatin, respectively. The analysis time for each sample was 1.8 min. The method was highly reproducible and gave peaks with excellent chromatography properties.     

Effect of silicon dioxide on expression of poly (ADP-Ribose) polymerase mRNA and protein

Silicon dioxide induces acute injury and chronic pulmonary fibrosis. International Agency for Research on Cancer (IARC) listed it as a human carcinogen in 1996. However, the molecular mechanisms to induce cancer are not understood yet. The content of poly (ADP-ribose) polymerases (PARP) mRNA and protein in Hela cells treated with concentrations of silicon dioxide up to 400 μg/ml was determined by real-time fluorogenetic quantitative PCR (RQ-PCR) and immunofluorescence assay, respectively. MTT assay was used to determine cell viability. The results showed that viability at 400 μg/ml silica was significantly decreased but not at lower concentrations. The protein content of γ-H2AX in silica-treated group was significantly higher than the controls. The PARP mRNA and protein levels were significantly reduced with a dose response manner from the lowest silicon dioxide level. Our findings suggested that silicon dioxide increased the expression of γ-H2AX and inhibited the expression of PARP mRNA and protein in Hela cells.