The Role of Ca<Superscript>2+</Superscript> Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca²⁺]_i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (?Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca²⁺]_i and ROS production, and reduced GSH content, catalase activity, ?Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca²⁺ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca²⁺ and drop in ?Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca²⁺ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca²⁺ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca²⁺-independent portion of oxidative stress and cytotoxicity emerges.     

Duloxetine Protects Human Neuroblastoma Cells from Oxidative Stress-Induced Cell Death Through Akt/Nrf-2/HO-1 Pathway

The contribution of oxidative stress to the pathophysiology of depression has been described in numerous studies. Particularly, an increased production of reactive oxygen species (ROS) caused by mitochondrial dysfunction can lead to neuronal cell death. Human neuroblastoma SH-SY5Y cells were used to investigate the neuroprotective effect of the antidepressant duloxetine against rotenone-induced oxidative stress. SH-SY5Y cells were pretreated with duloxetine (1–5 µM) for 24 h followed by a 24-h rotenone exposure (10 µM). The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) inhibitor LY294002 (10 µM) and the heme oxygenase 1 (HO-1) inhibitor zinc protoporphyrin IX-ZnPP (5 µM) were added to cultures 1 h prior duloxetine treatments. After treatments cell viability and ROS generation were assessed. NF-E2-related factor-2 (Nrf2) nuclear translocation was assessed by immunofluorescent staining after 4 and 8 h of duloxetine incubation. Furthermore, the Nrf2 and HO-1 mRNA expression was carried out after 4–48 h of duloxetine treatment by qRT-PCR. Duloxetine pretreatment antagonized rotenone-induced overproduction of ROS and cell death in SH-SY5Y cells. In addition, a 1-h pretreatment with LY294002 abolished duloxetine’s protective effect. Duloxetine also induced nuclear translocation of the Nrf2 and the expression of its target gene, HO-1. Finally, the HO-1 inhibitor, ZnPP, suppressed the duloxetine protective effect. Overall, these results indicate that the mechanism of duloxetine neuroprotective action against oxidative stress and cell death might rely on the Akt/Nrf2/HO-1 pathways.     

Antiproliferative and cytotoxic effects of purple pitanga (Eugenia uniflora L.) extract on activated hepatic stellate cells

The presence of phenolic compounds in fruit‐ and vegetable‐rich diets has attracted researchers' attention due to their health‐promoting effects. The objective of this study was to evaluate the effects of purple pitanga (Eugenia uniflora L.) extract on cell proliferation, viability, mitochondrial membrane potential, cell death and cell cycle in murine activated hepatic stellate cells (GRX). Cell viability by 3‐(4,5‐dimethylthiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay was significantly decreased on cells treated with 50 and 100 µg ml^?1 of purple pitanga extract for 48 and 72 h, and the percentage of dead cell stained with 7‐amino‐actinomycin D was significantly higher in treated cells. The reduction of cell proliferation was dose dependent, and we also observed alterations on cell cycle progression. At all times studied, GRX cells treated with 50 and 100 µg ml^?1 of purple pitanga showed a significant reduction in cellular mitochondrial content as well as a decrease in mitochondrial membrane potential. Furthermore, our results indicated that purple pitanga extract induces early and late apoptosis/necrosis and necrotic death in GRX cells. This is the first report describing the antiproliferative, cytotoxic and apoptotic activity for E. uniflora fruits in hepatic stellate cells. The present study provides a foundation for the prevention and treatment of liver fibrosis, and more studies will be carried to elucidate this effect. Copyright © 2013 John Wiley & Sons, Ltd.     

Short-term inhibition of prolactin secretion by naloxone treatment in the pregnant gilt

The involvement of endogenous opioids in modulation of prolactin (PRL) secretion during pregnancy in the pig was studied. Twenty-four crossbred pregnant gilts (150 ± 10 kg) were cannulated via the cephalic vein 24–48 h before treatment with 1 mg kg⁻¹ body weight of naloxone (NAL) or 3 ml of saline (CONT) i.v. at Day 40 (NAL, n = 6; CONT, n = 6) or Day 70 (NAL, n = 6; CONT, n = 6) of pregnancy. Blood plasma was collected at 15 min intervals from 1 h before to 3 h after treatment with NAL or saline. At Day 40 of pregnancy, administration of NAL caused a decrease in mean plasma PRL concentrations at 60 min, 120 min and 180 min post-treatment (NAL, 19.1 ± 1.3 ng ml⁻¹, P < 0.05; 15.8 ± 0.6 ng ml⁻¹, P < 0.001; 14.6 ± 0.7 ng ml⁻¹, P < 0.001, respectively) when compared with the CONT group (22.9 ± 0.7 ng ml⁻¹, 21.6 ± 0.6 ng ml⁻¹ and 22.4 ± 0.5 ng ml⁻¹, respectively). Mean plasma estradiol concentration was higher (P < 0.01) in the NAL group during the second and third hour post-treatment than in the CONT group. At Day 70 of pregnancy, infusion of NAL also decreased (P < 0.001) plasma PRL concentrations at 60 min, 120 min and 180 min after treatment (20.1 ± 1.6 ng ml⁻¹, 16.2 ± 1.5 ng ml⁻¹ and 14.8 ± 0.4 ng ml⁻¹, respectively) compared with the CONT group (33.4 ± 1.7 ng ml⁻¹, 34.1 ± 1.3 ng ml⁻¹ and 29.1 ± 0.9 ng ml⁻¹, respectively). Estradiol concentrations were not different (P > 0.05) between groups in this stage of gestation. Mean concentrations of progesterone were similar during the pre- and post-treatment periods in both stages of pregnancy.These data would suggest a possible role of the opioids in modulation of PRL secretion at these stages of pregnancy in the pig.     

Dual functional nisin-multi-walled carbon nanotubes coated filters for bacterial capture and inactivation

Background

Removal of pathogens from water is one way to prevent waterborne illness. In this paper, we developed dual functional carbon nanotube (CNT) modified filters for bacterial capture and inactivation, utilizing multi-walled CNTs (MWCNTs) to coat on commercially available filters and making use of the exceptional adsorption property of CNTs to adsorb a natural antimicrobial peptide-nisin on it. Two types of MWCNTs with different outer layer diameters were used (MWCNTs1: <8 nm in diameter; MWCNTs2: 10–20 nm in diameter).

Results

The thickness of MWCNT layers, surface morphology, and surface hydrophobicity of both types of MWCNT coated filters were characterized. The MWCNT coating on filters significantly increased the surface hydrophobicity. The absorption of nisin and the capture of bacterial pathogens were correlated with increased surface hydrophobicity. The MWCNTs1 and MWCNTs2 filters with 1.5 mg MWCNTs loading captured 2.44 and 3.88 log of cells, respectively, from aqueous solutions containing a total of ~10⁶ CFU/mL cells. Nisin deposit at the amount of 0.5 mg on the surfaces of MWCNT filters significantly reduced the viability of captured B. anthracis cells by 95.71–97.19 %, and inhibited the metabolic activities of the captured cells by approximately 98.3 %.

Conclusions

The results demonstrated that the MWCNT-nisin filters achieved dual functions in bacterial pathogen capture and inhibition in one single filtration step, which is potentially applicable in removing undesired microorganisms from water sources and inhibiting captured Gram positive bacteria activities.

     

Study of hepatotoxicity and oxidative stress in male Swiss-Webster mice exposed to functionalized multi-walled carbon nanotubes

Carbon nanotubes (CNTs), the most promising material with unique characteristics, find its application in different fields ranging from composite materials to medicine and from electronics to energy storage. However, little is known about the mechanisms behind the interaction of these particles with cells and their toxicity. The aim of this study was to assess the effects, after intraperitoneal (ip) injection, of functionalized multi-walled carbon nanotubes (MWCNT) (carboxyl groups) on various hepatotoxicity and oxidative stress biomarkers (ROS, LHP, ALT, AST, ALP, and morphology of liver) in the mouse model. The mice were dosed ip at 0.25, 0.5, and 0.75 mg/kg/day for 5 days of purified/functionalized MWCNTs and two controls (negative; saline and positive; carbon black 0.75 mg/kg) as appropriate. Samples were collected 24 h after the fifth day treatment following standard protocols. Exposure to carboxylated functionalized MWCNT; the body-weight gain of the mice decreased, induced reactive oxygen species (ROS), and enhanced the activities of serum amino-transferases (ALT/AST), alkaline phosphatases (ALP), and concentration of lipid hydro peroxide compared to control. Histopathology of exposed liver showed a statistically significant effect in the morphological alterations of the tissue compared to controls. The cellular findings reported here do suggest that purified carboxylated functionalized MWCNT has the potential to induce hepatotoxicity in Swiss-Webster mice through activation of the mechanisms of oxidative stress, which warrant in vivo animal exposure studies. However, more studies of functionalization in the in vivo toxicity of MWCNTs are required and parallel comparison is preferred.     

Induction of apoptosis in rat lung epithelial cells by multiwalled carbon nanotubes

Carbon nanotubes (CNTs), the most promising material with unique characteristics, find its application in different fields ranging from composite materials to medicine and from electronics to energy storage. However, little is known about the mechanism behind the interaction of these particles with cells and their toxicity. So, here we investigated the adverse effects of multiwalled CNTs (MWCNTs) in rat lung epithelial (LE) cells. The results showed that the incubation of LE cells with 0.5–10 μg/mL of MWCNTs caused a dose‐ and time‐dependent increase in the formation of free radicals, the accumulation of peroxidative products, the loss of cell viability, and antioxidant depletion. The significant amount of incorporation of dUTPs in the nucleus after 24 h confirms the induction of apoptosis. It was also observed that there is an increase in the activity of both caspases‐3 and caspase‐8 in cells, with increases in time and the concentration of MWCNTs. No significant incorporation of dUTPs was observed in cells, incubated with z‐VAD‐fmk , which confirmed the role of caspases in DNA fragmentation. The present study reveals that MWCNTs induced oxidative stress and stimulated apoptosis signaling pathway through caspase activation in rat LE cell lines. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:333–344, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20296     

Effect of addition of buserelin acetate to the extender on motility and viability of bovine spermatozoa

The present study was aimed to determine the effect of GnRH analog (buserelin acetate) on the quality of bovine spermatozoa stored at 16°?C for 24?h. Semen collected in the summer season from June to September from healthy Polish Holstein–Friesian bulls. Ejaculates were centrifuged, divided and diluted to the final concentration of 240?×?10⁶ spermatozoa/mL using animal protein–free commercial BIOXcell^® extender (IMV Technologies, L’aigle, France) (Control) or with BIOXcell^® extender supplemented with buserelin acetate and stored 0, 8 and 24?h. Sperm motility parameters analysis was performed using a computer-assisted sperm analysis (CASA) system. The viability of spermatozoa was performed using flow cytometer. The addition of buserelin acetate to BIOXcell^® extender did positively affect the total motility (was higher in the observed samples with the addition of 2?µg/mL and 4?µg/mL than in the control group), progressive motile (forward progressing sperm was significantly increased (p?<?0.05) over the control group at the 0?h and 8?h of incubation following the supplementation of 2, 4 and 8?μg/mL buserelin acetate) and viability of spermatozoa (the number of live spermatozoa was significantly higher (p?<?0.05) in 2?µg/mL and 4?µg/mL samples with buserelin acetate at 8th hour of incubation and in sample with 4?µg/mL at 24th hour of incubation compared to the control group). We recommend adding 4?µg/mL to the extender to improve the quality of bovine semen.     

Optimization of indole acetic acid production by isolated bacteria from Stevia rebaudiana rhizosphere and its effects on plant growth

The ability to synthesize Indole-3-acetic acid (IAA) is widely associated with the plant growth promoting rhizobacteria (PGPR). The present work deals with isolation and characterization of such bacteria from the rhizosphere of medicinal plant Stevia rebaudiana and optimization of IAA production from its isolates. The optimization of IAA production was carried out at different pH and temperature with varied carbon and nitrogen sources of culture media. Out of different isolates obtained, three of them were screened as efficient PGPRs on the basis of different plant growth promoting attributes. Isolates CA1001 and CA2004 showed better production of IAA at pH 9 (91.7?µg?ml^?1) and at temperature 37?°C (81.7?µg?ml^?1). Dextrose (1%) was found to be the best carbon source for isolate CA1001 with 104?µg?ml^?1 IAA production. Isolate CA 2004 showed best production of IAA 36?µg?ml^?1 and 34?µg?ml^?1 at 1.5% and 1% Beef extract as nitrogen source respectively. Isolate CA 1001 showed 32?µg?ml^?1 IAA production at 0.5% nicotinic acid concentration. From the current study, CA1001 and CA2004 emerged as noble alternatives for IAA production further which also resulted in root and shoot biomass generation in crop plants, hence can be further used as bio-inoculants for plant growth promotion.     

Antimicrobial activity and cytotoxicity trait of a bioactive peptide purified from Lactococcus garvieae subsp. bovis BSN307T

A bioactive peptide of 8595 Da was purified from the cell free supernatant of Lactococcus garvieae subsp. bovis BSN307^T. MALDI MS/MS peptide mapping and the data base search displayed no significant similarity to any reported antimicrobial peptide of LAB. This peptide at a dose concentration of 200 µg ml⁻¹ inhibited the growth of both Gram-positive and Gram-negative bacteria by 58–89% and a dose of 500 µg ml⁻¹ scavenged 50% of DPPH-free radicals generated. Interestingly, cytotoxicity assay demonstrated that 17 µg ml⁻¹ of peptide selectively inhibited 50% proliferation of mammalian cancer cell lines HeLa and MCF-7 whereas normal H9c2 cells remained unaffected. Fluorescent microscopic analysis after DAPI nuclear staining of HeLa cells showed characteristics of apoptosis and activation of caspase-3 was ascertained by caspase-3 fluorescence assay.     

Piperine,an active ingredient of white pepper,suppresses the growth of multidrug-resistant toxigenic Vibrio cholerae and other pathogenic bacteria

Emergence and rapid spread of multidrug-resistant (MDR) bacteria including Vibrio cholerae are a global public health issue. Much attention has been paid to natural compounds, such as spices and herbs to find novel antimicrobial compounds as they are considered to be cheaper alternatives to develop as a drug. Here, we show that methanol extract of white pepper could inhibit the growth of V. cholerae O1 El Tor variant, responsible for the recent outbreaks/epidemics. Furthermore, we demonstrate for the first time that piperine, the major component of white pepper, showed a dose-dependent bactericidal effect on V. cholerae growth irrespective of their biotypes and serogroups in the presence of 200 and 300 µg ml⁻¹ of piperine, respectively. Piperine also inhibited the growth of MDR strains of Pseudomonas aeruginosa, Escherichia coli isolated from poultry and enterohemorrhagic/enteroaggregative E. coli O104 in the presence of 200 µg ml⁻¹. Interestingly, we did not observe any significant inhibitory effect of piperine on E. coli strains isolated from healthy person even up to 200 µg ml⁻¹. Our data suggest that piperine could be a novel antimicrobial agent in therapeutic and preventive applications against infections caused by pathogenic bacteria including MDR strains.     

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux ‘signatures’ during these stress conditions are poorly characterized. We investigated the kinetics of K⁺, Ca²⁺ and H⁺ ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non‐invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K⁺ efflux was observed following acute ER stress with peak K⁺ efflux being ?30·6 and ?138·7 nmolm^?2 s^?1 for 10 and 50 µg ml^?1, respectively (p < 0·01). TM‐dependent Ca²⁺ uptake was more prolonged with peak values of 0·85 and 2·68 nmol m^?2 s^?1 for 10 and 50 µg ml^?1 TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K⁺ efflux and Ca²⁺ uptake. While K⁺ changes were significantly higher at each time point tested, Ca²⁺ uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K⁺ efflux and Ca²⁺ uptake. Functional assays to investigate the effect of inhibiting K⁺ efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K⁺, Ca²⁺ and H⁺ ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

Antibiofilm and antifungal activities of medium-chain fatty acids against Candida albicans via mimicking of the quorum-sensing molecule farnesol

Candida biofilms are tolerant to conventional antifungal therapeutics and the host immune system. The transition of yeast cells to hyphae is considered a key step in C. albicans biofilm development, and this transition is inhibited by the quorum-sensing molecule farnesol. We hypothesized that fatty acids mimicking farnesol might influence hyphal and biofilm formation by C. albicans. Among 31 saturated and unsaturated fatty acids, six medium-chain saturated fatty acids, that is, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and lauric acid, effectively inhibited C. albicans biofilm formation by more than 75% at 2 µg ml⁻¹ with MICs in the range 100–200 µg ml⁻¹. These six fatty acids at 2 µg ml⁻¹ and farnesol at 100 µg ml⁻¹ inhibited hyphal growth and cell aggregation. The addition of fatty acids to C. albicans cultures decreased the productions of farnesol and sterols. Furthermore, down-regulation of several hyphal and biofilm-related genes caused by heptanoic or nonanoic acid closely resembled the changes caused by farnesol. In addition, nonanoic acid, the most effective compound diminished C. albicans virulence in a Caenorhabditis elegans model. Our results suggest that medium-chain fatty acids inhibit more effectively hyphal growth and biofilm formation than farnesol.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Synergistic effect on anti-methicillin-resistant Staphylococcus aureus among combinations of α-mangostin-rich extract,lawsone methyl ether and ampicillin

α-Mangostin-rich extract (AME) exhibited satisfactory inhibitory activities against all tested MRSA strains, with minimum inhibitory concentrations (MICs) of 7·8–31·25 µg ml⁻¹, whereas lawsone methyl ether (LME) and ampicillin revealed weak antibacterial activity with MICs of 62·5–125 µg ml⁻¹. However, the combination of AME and LME showed synergistic effects against all tested MRSA strains with fractional inhibitory concentration index (FICI) values of 0·008–0·009, while the combination of AME and ampicillin, as well as LME and ampicillin produced synergistic effects with FICIs of 0·016–0·257. A time-kill assay against MRSA (DMST 20654 strain) revealed a 6-log reduction in CFU per ml, which completely inhibited bacterial growth for the combinations of AME and LME, AME and ampicillin, and LME and ampicillin at a 8-h incubation, while those against MRSA (2468 strain) were at 10-h incubation. The combination of α-mangostin and LME as well as the combinations of each compound with ampicillin synergized the alteration of membrane permeability. In addition, α-mangostin, LME and ampicillin inhibited the biofilm formation of MRSA. These findings indicated that the combinations of AME and LME or each of them in combination with ampicillin had enhanced antibacterial activity against MRSA. Therefore, these compounds might be used as the antibacterial cocktails for treatment of MRSA.     

Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture

The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP - 1, 2.5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST 1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS production was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation.     

Bronchipret® syrup containing thyme and ivy extracts suppresses bronchoalveolar inflammation and goblet cell hyperplasia in experimental bronchoalveolitis

Background/purposeAcute bronchitis (AB) is a common lung condition characterized by inflammation of the large bronchi in response to infection. Bronchipret^® syrup (BRO), a fixed combination of thyme and ivy extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to identify potential mechanisms of action.MethodsBronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o. once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24–72 h post LPS challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO).ResultsBRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular release of LTB₄ (IC₅₀ = 36 µg⋅ml⁻¹) and cysLT (IC₅₀ = 10 µg⋅ml⁻¹) and inhibited the activity of isolated 5-LO (IC₅₀ = 19 µg⋅ml⁻¹).ConclusionBRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may contribute to its observed clinical efficacy in AB.     

Differential protein expression due to Se deficiency and Se toxicity in rat liver

There is a U-shaped dose-response between selenium (Se) status and health outcomes, but underlying metabolic processes are unclear. This study aims to identify candidate proteins in liver regulated by dietary Se, ranging from deficiency to toxic. Male rats (n=4) were fed graded Se concentrations as selenite for 28 days. Bulk Se analysis was performed by ICP-MS on both soluble and insoluble fractions. Soluble fraction samples were chromatographically separated for identification of selenocompounds by SEC-ICP-MS and protein quantification by LC-MS/MS. Bioinformatics analysis compared low-Se (0 and 0.08 µg Se g⁻¹) and high-Se (0.8, 2 and 5 µg Se g⁻¹) with adequate-Se (0.24 µg Se g⁻¹) diets. Major breakpoints for Se were seen at 0.8 and 2 µg Se g⁻¹ in the insoluble and soluble fractions, respectively. Glutathione peroxidase 1 protein abundance reached a plateau at ≥0.08 µg Se g⁻¹diet; Se bound to selenium binding protein 2 was observed with 2 and 5 µg Se g⁻¹ Se. The extreme diets presented the highest number of differentially expressed (P value <0.05, FC ≥1.2) proteins in comparison to the adequate-Se diet (0 µg Se g⁻¹: 45 proteins; 5 µg Se g⁻¹: 59 proteins); 13 proteins were commonly affected in 0 and 5 µg Se g⁻¹ treatments. Network analysis revealed that the metabolism of glutathione, xenobiotics and amino acids were enriched in both 0 and 5 µg Se g⁻¹ diets, indicating a U-shape effect of Se. This similarity is likely due to down-stream effects of lack of essential selenoproteins in Se deficiency and due to toxic effects of Se that exceeds the capacity to cope with excess Se.     

Removal of two pathogenic scuticociliates Miamiensis avidus and Miamiensis sp. using cells or culture filtrates of the dinoflagellate Alexandrium andersonii

Scuticociliatosis, which is caused by parasitic protistan pathogens known as scuticociliates, is one of the most serious diseases in marine aquaculture worldwide. Thus, elimination of these ciliates is a primary concern for scientists and managers in the aquaculture industry. To date, formalin and other toxic chemicals have been used as anti-scuticociliate agents, but issues regarding their secondary effects often arise. Consequently, development of safer methods is necessary. To find out a safe method of controlling scuticociliate populations in aqua-tanks or small-scale natural environments, cultures of 14 phototrophic dinoflagellates were tested to determine whether they were able to control populations of the common scuticociliates Miamiensis avidus and Miamiensis sp. isolated from Korean waters. Among the dinoflagellates tested, both cells and culture filtrates of Alexandrium andersonii effectively killed M. avidus and Miamiensis sp. The minimal concentration of cells and equivalent culture filtrates of A. andersonii to kill all M. avidus cells within 48 h of incubation was ca. 2500 and 4500 cells ml⁻¹, respectively; whereas those needed to kill all Miamiensis sp. cells were ca. 1000 and 4500 cells ml⁻¹, respectively. It was estimated that 1 m³ of the stock culture containing 20,000 A. andersonii cells ml⁻¹ could eliminate all M. avidus cells in 7 m³ of waters within the aqua-tanks on land and all Miamiensis sp. cells in 19 m³ of waters within 48 h. None of the brine shrimp Artemia salina nauplii incubated with concentrations of 50–4500 A. andersonii cells ml⁻¹ for 24 h was dead. Furthermore, none of the flounder Paralichthys olivaceus juveniles incubated with a mean concentration of ca. 2280 A. andersonii cells ml⁻¹ for 96 h was dead. Therefore, A. andersonii cultures may be used as a safe biological method for controlling populations of scuticociliates and can replace toxic formalin. The results of this study provided the basis for developing the method to control scuticociliate populations and understanding interactions between scuticociliates and phototrophic dinoflagellates in marine ecosystems.     

High synergistic antibacterial,antibiofilm, antidiabetic and antimetabolic activity of Withania somnifera leaf extract-assisted zinc oxide nanoparticle

Nanotechnology is currently gaining immense attention to combat food borne bacteria, and biofilm. Diabetes is a common metabolic disease affecting majority of people. A better therapy relies on phytomediated nanoparticle synthesis. In this study, W. somnifera leaf extract-assisted ZnO NPs (Ws-ZnO NPs) was synthesized and characterized. From HR-TEM analysis, it has been found that the hexagonal wurtzite particle is 15.6 nm in size and − 12.14 mV of zeta potential. A greater antibacterial effect of Ws-ZnO NPs was noticed against E. faecalis and S. aureus at 100 µg mL⁻¹. Also, the biofilm of E. faecalis and S. aureus was greatly inhibited at 100 µg mL⁻¹ compared to E. coli and P. aeruginosa. The activity of α-amylase and α-glucosidase enzyme was inhibited at 100 µg mL⁻¹ demonstrating its antidiabetic potential. The larval and pupal development was delayed at 25 µg mL⁻¹ of Ws-ZnO NPs. A complete mortality (100%) was recorded at 25 µg mL⁻¹. Ws-ZnO NPs showed least LC₅₀ value (9.65 µg mL⁻¹) compared to the uncoated ZnO NPs (38.8 µg mL⁻¹) and leaf extract (13.06 µg mL⁻¹). Therefore, it is concluded that Ws-ZnO NPs are promising to be used as effective antimicrobials, antidiabetic and insecticides to combat storage pests.