Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5–11) and temperature (25–70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca2+ and Mg2+. EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level.The present study describes the isolation of a new protease producing  Streptomyces species are the most industrially useful because of their capacity of producing numerous secondary metabolites, particularly antibiotics. Similarly, these bacteria offer a second industrial interesting use by producing large amounts of proteolytic enzymes, with different substrate specificities [Actinomycetes, a Gram-positive filamentous bacteria, can degrade various macromolecules in soil . Among aficities . The invficities . Unlike ficities . Still,  Streptomyces is determined by the taxonomic position of the producers [ Streptomyces for producing proteases is justified by their ability to release the proteins into extracellular media. Such a capability is generally regarded as safe (GRAS) with food and drug administration. Streptomyces spp. that produce proteases include S. clavuligerus, S. griseus, S. rimouses, S. thermoviolaceus, and S. thermovulgaris [ Streptomyces griseus [ Streptomyces fradiae [ Streptomyces like Streptomyces clavuligerus, Streptomyces gulbargensis, Streptomyces viridifaceins, and Streptomyces sp. [The composition of the protease complexes secreted byroducers –8. The pvulgaris . Some of griseus  and Stre fradiae , have beyces sp. –13. Streptomyces flavogriseus HS1 strain, isolated from a Tunisian soil. We report the biochemical characterization of the crude enzyme for evaluation of its biotechnological potential as detergent additive.Present study describes the isolation of a multiple protease producing3 2, NaCl 2, K2HPO4 2, MgSO4·7H2O 0.05, CaCO3 0.02, FeSO4·7H2O 0.01, and 15 agar [The isolation of the Actinomycete strains from a soil sample was done by serial dilution plate technique on ISP4 agar media containing (g/L) starch 10, casein 0.3, KNO 15 agar .The 16S rRNA gene of the HS1 strain was amplified by PCR using the following primers: F (forward), 5′-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3′ and R (reverse), 5′-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3′. The PCR mixture contained 30 pmol of primers, 20 pmol of each deoxynucleoside triphosphate, polymerisation buffer, and 5 U Taq polymerase. The PCR program involved 35 cycles of denaturing at 94°C for 1 min, primer annealing at 55°C for 1 min, and extension at 72°C for 90 s. The sequencing was performed three times using the DNA sequencer ABI PRISM 3100/3100-Avant Genetic Analyser . 16S rDNA sequence was searched for similarities to known sequences in the GenBank database  using the BLAST search program. The sequence was aligned with those of the reference strains using ClustalW . A phyloμg of tyrosine per minute under the experimental conditions used. All measurements were carried out in triplicate.Measuring of the protease activity was done as described by Kembhavi et al. , using c2PO4 1.0, K2HPO4 3.4, MgSO4·7H2O 0.3, FeSO4·7H2O 0.01, ZnCl2 0.1, CuSO4·7H2O 0.01, MgCl2·4H2O 0.003, CaCl2 0.01, NaCl 0.03, pH 7.0 [The proteolytic isolates were cultured in three different liquid medium: FermII media: (g/L) dextrin 20, tryptone 10, KH, pH 7.0 , gelatin, pH 7.0 .4 to 106 spores/mL) was added to a 250 mL Erlenmeyer flask containing 100 mL of liquid media [A 1 mL spore suspension .2HPO4·NaOH, pH 12.0.Protease activity was assayed over the pH range 5.0–12.0 at 50°C for 10 min, using casein as a substrate. The effect of pH on enzyme stability was evaluated by measuring the residual enzyme activity after incubation at various pH for 60 min at 25°C. The following buffer systems were used: 100 mM glycine-HCl, pH 4.0 and 5.0; 100 mM sodium acetate, pH 6.0; 100 mM phosphate-buffer, pH 7.0; 100 mM Tris-HCl, pH 8.0; 100 mM glycine-NaOH, pH 9.0 and 10; and 100 mM Na+ or K+) or divalent  metal ions, at a concentration of 5 mM, on protease activity were investigated by adding them to the reaction mixture. The activity in the absence of any additives was taken as 100%.The effects of different monovalent  and oxidizing agents (sodium perborate) on enzyme stability were studied by preincubating the crude enzyme for 1 h at 25°C. The residual activity was measured at pH 7.0 and 50°C. The activity of the enzyme without any additive was taken as 100%. Streptomyces flavogriseus HS1 were incubated with different diluted detergents (1/100) for 1 h at 30°C, 40°C, and 50°C and then the remaining activities were determined under the standard assay conditions. The enzyme activity of a control, without detergent, incubated under similar conditions, was taken as 100%.The compatibility of the HS1 extracellular proteases with commercial laundry detergents was studied using Ariel , Newdet , and Dixan  as solid detergents. The endogenous proteases contained in these detergents were inactivated by heating the diluted detergents for 1 h at 65°C prior to the addition of the enzyme preparation. The extracellular proteases ofP < 0.05. Results represent the means of at least two determination carried out in duplicate. The difference between values did not exceed 5%.Statistical analyses were performed with Statgraphics ver. 5.1, professional edition  using ANOVA analysis. Differences were considered significant at  Streptomyces since it possessed nonfragmented substrate mycelia, aerial hyphae, and smooth spores organized in straight chains. Analysis of the 16S rRNA gene sequence of this strain showed a high similarity (100%) with Streptomyces flavogriseus (Samples were taken from an organic rich soil in Sfax city (Tunisia). Isolation of the actinomycete strains was obtained after 96 h of incubation at 30°C. One isolate was selected for further studies because of its important extracellular proteases secretion and named HS1 strain . HS1 strogriseus .Streptomyces flavogriseus was well known to produce several enzymes such as cellulose, xylanase, and glucose isomerase [somerase –21, but  Streptomyces were tested, among them are FermII [ Streptomyces flavogriseus HS1 extracellular proteases ( Streptomyces flavogriseus HS1 (data not shown) indicates that the maximum protease activity (99 U/mL) was obtained after 96 h of cultivation, when cells were in the stationary phase, and its production was not growth associated. Similar results were obtained by Gibb and Strohl [ Streptomyces peucetius occurred after 100 h of cultivation at the stationary phase growth. This period was shorter than that of the well-studied Streptomyces  after 48 h of growth.Three liquid culture media optimized for the production of extracellular proteases ine FermII , ISP4 [1e FermII , and gelroteases . The ferd Strohl , who obsoduction  showed t Streptomyces flavogriseus HS1 showed at least five major proteases. This is a common feature for the streptomycetes [To give more information about the diversity of extracellular proteases secreted by HS1 strain, zymogram analysis was done as described in the “Material and Methods” section. As shown in omycetes , 23. Howomycetes . All theomycetes . Streptomyces flavogriseus HS1 is shown in  Streptomyces strains in the literature with optimum pH range being between 6.0 and 12.0 [ Streptomyces flavogriseus proteases was similar to that from other Streptomyces species, such as Streptomyces griseus pronase [ Streptomyces described proteases, showing maximum activity at pH 8.0 like Streptomyces sp. DP2 [ Streptomyces sp. CN902 [The pH profile of protease activity fromand 12.0 . The opt pronase . Therefo sp. DP2  and Strep. CN902 . The pH  Streptomyces flavogriseus HS1 is presented in  Thermoactinomyces vulgaris and Nocardiopsis dassonvillei, S. corchorusii [ S. megasporus [ S. thermovulgaris [ S. thermoviolaceus [ Streptomyces flavogriseus HS1 rapidly fell, as shown for proteases of Streptomyces spp. and other bacteria.The temperature profile of protease activity fromchorusii , S. megagasporus , S. thervulgaris , and S. iolaceus . Above 6 Streptomyces flavogriseus HS1 proteases were stable at 40 and 50°C after 1 h incubation. At low temperatures (−20 and 4°C), the crude enzyme preparation retained 75% of its activity after 2 months. El-Raheem et al. [ S. corchorusii, activity did not decrease after storage at −20°C for one year, at pH values between 4.0 and 12.0, and repeated freezing and thawing.The thermal stability profile of the crude enzyme showed a high stability at temperatures below 40°C but was inactivated at higher temperatures . After 4m et al.  observed Streptomyces flavogriseus HS1 crude enzyme were studied at pH 7.0 and 50°C by the addition of the respective cations to the reaction mixture  and the removal of Ca2+ from the strong binding site is associated with a significant reduction in thermal stability [Proteases can be classified by their sensitivity to various inhibitors . In ordetability . From th Streptomyces flavogriseus HS1 proteases were less stable against the strong anionic surfactant (SDS) and retained only 19% of its activity in the presence of 0.1% (w/v) SDS. However, HS1 protease activity was little influenced by oxidizing agents and retained 64.1% and 39.8% of its activity after incubation for 1 h at 25°C in the presence of 0.1% and 1% sodium perborate, respectively  (The high activity and stability of thend 50°C) . The dat Bacillus sp. SSR1 retained 37% of its initial activity after 1 h incubation at 40°C in the presence of Ariel at a concentration of 5 mg/mL. Alkaline protease from Conidiobolus coronatus retained only 16% activity in Revel, 11.4% activity in Ariel, and 6.6% activity in Wheel at 50°C, in the presence of 25 mM CaCl2 [Singh et al.  reportedmM CaCl2 . Streptomyces flavogriseus HS1, which produces at least five proteases as described by zymogram technique. Crude protease was shown to have optimum activity at pH 7 and 50°C. The crude enzyme has a good stability toward the oxidizing agents and was found to be useful as a detergent additive.This work describes the isolation of an actinomycete strain identified as


Polypedilum vanderplanki, accumulate very large amounts of trehalose as a compatible solute on desiccation, but the molecular mechanisms underlying this accumulation are unclear. We therefore isolated the genes coding for trehalose metabolism enzymes, i.e. trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) for the synthesis step, and trehalase (TREH) for the degradation step. Although computational prediction indicated that the alternative splicing variants (PvTpsα/β) obtained encoded probable functional motifs consisting of a typical consensus domain of TPS and a conserved sequence of TPP, PvTpsα did not exert activity as TPP, but only as TPS. Instead, a distinct gene (PvTpp) obtained expressed TPP activity. Previous reports have suggested that insect TPS is, exceptionally, a bifunctional enzyme governing both TPS and TPP. In this article, we propose that TPS and TPP activities in insects can be attributed to discrete genes. The translated product of the TREH ortholog (PvTreh) certainly degraded trehalose to glucose. Trehalose was synthesized abundantly, consistent with increased activities of TPS and TPP and suppressed TREH activity. These results show that trehalose accumulation observed during anhydrobiosis induction in desiccating larvae can be attributed to the activation of the trehalose synthetic pathway and to the depression of trehalose hydrolysis.Larvae of an anhydrobiotic insect,  Polypedilum vanderplanki, can withstand drought stress by the induction of an ametabolic state termed ‘cryptobiosis’ or ‘anhydrobiosis’ dATP using a Strip-EZ labeling kit . The hybridized blot was analyzed by BAS 2500 .Total RNA was isolated from dehydrating larvae using TRIzol . Northern blot analysis was performed as described previously ,33. Brieg for 10 min at room temperature, and the supernatants were collected. The concentration of protein was determined as described previously methionine . The reaction products were separated by 15% SDS/PAGE, and the gel was applied to western blot analyses as described above, or for autoradiography to confirm protein synthesis.m potassium-phosphate buffer (pH 6.8), containing 0.1 mm EDTA, 15 mm MgCl2, 4 μm glucose-1,6-bisphosphate, 0.1 U phosphoglucomutase, 0.6 U glucose-6-phosphate dehydrogenase, 2 mg·mL−1 glycogen, 0.4 mm NADP and 10 μL of protein extract, were incubated at 30 °C for 30 min, monitoring the change in the absorbance at 340 nm (A340). Because the inactive form of GP is activated by an allosteric effector, such as AMP, to determine total GP (active ‘a’ form and inactive ‘b’ form) activity, the reactions were performed in the presence of an additional 1 mm 5′-AMP.GP (EC 2.4.1.1) assays were performed as follows: 100 μL of 45 mm glucose-6-phosphate, 2.5 mm UDP-glucose, 2.5 mm MgCl2, 100 mm KCl, 1.25 mm phosphoenolpyruvate, 20 μL pyruvate kinase/lactate dehydrogenase (34 μL·mL−1), 0.3 mm NADH, 30 mm Tris/HCl (pH 7.4) and 5 μL of protein extract, were incubated at 30 °C for 30 min, monitoring the change in A340 that depends on NADH oxidation. In the case of samples from in vitro transcription and translation, 1.2 μL each of the products were incubated at 30 °C for 2 h, and then at 95 °C for 10 min to stop the reaction.For TPS assays, 200 μL of reaction mixture, containing 2.5 mm trehalose-6-phosphate, 2.5 mm MgCl2, 30 mm Tris/HCl (pH 7.4) and 20 μL of protein extract. In assays for the in vitro transcription and translation products, 1.2 μL of each of the preparations was used. The mixtures were incubated at 30 °C for 1 h, and then at 95 °C for 10 min to stop the reaction. The reaction product  was measured by HPLC [Assays for TPP activity were performed in 200 μL of reaction mixture containing 2.5 m by HPLC .m phosphate buffer (pH 6.0) containing 20 mm trehalose and an appropriate amount of protein preparation. After incubation at 30 °C for 0.5–1 h, the reaction mixture was boiled for 5 min. As a control, another reaction mixture was immediately boiled without incubation. The reaction products  were measured by HPLC [TREH activity was assayed in 250 μL of 15 m by HPLC .in vivo in completely desiccated larvae [A desiccation treatment of 48 h is required to completely desiccate larvae under laboratory conditions ,41. Enzyd larvae .S. cerevisiae deletion mutants were purchased from Open Biosystems, Huntsville, AL. The deletion strains for TPS1  and TPS2  were transformed with pUG35 , which contains the ORF of PvTpsα, PvTpsβ and PvTpp under the MET25 promoter [GFP ORF instead of the target genes. After selection on synthetic defined (SD) medium lacking uracil, transformants were confirmed by colony PCR. Three independent colonies were picked up for each strain. For the complementation test of the tps1 mutant, transformants of the tps1 deletion mutant with PvTpsα and PvTpsβ were grown in SD medium containing 2% galactose and lacking uracil and methionine at 30 °C to an exponential phase. After harvesting of the yeast cells, a dilution series of 104–101 cells was prepared, and each solution was spotted onto yeast extract and peptone (YP) medium containing galactose conditioned in hyperosmolarity with 1 m NaCl, 50% sucrose or 1.5 m sorbitol. For complementation tests of the tps2 mutant, diluted series of transformants of the tps2 deletion mutant with PvTpp were prepared as for tps1. Each cell suspension was spotted onto SD medium containing 2% galactose and lacking methionine and uracil. To confirm the rescue of the temperature sensitivity of the tps2Δ mutant, the plates were incubated at 45 °C for 5 h and then at 30 °C for 3–4 days.The promoter . For theet al. [PvTpsα- or PvTpsβ-introduced yeast strains were cultured in SD medium containing galactose and lacking uracil and methionine at 30 °C for 48 h until the growth curve entered the stationary phase. Yeast cells were harvested and homogenized with glass beads in 80% ethanol. After centrifugation at 20 000 g for 30 min, the supernatants were collected and subjected to sample preparation for HPLC analysis [The amount of trehalose was determined by HPLC according to Watanabe et al. . For theanalysis .


To characterize Ca2+ signaling in striatal cells, spontaneous cytoplasmic Ca2+ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca2+ transients (referred to as slow Ca2+ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca2+ oscillation. Depletion of the intracellular Ca2+ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca2+ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca2+ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca2+ oscillation in both putative-neurons and astrocytes. The slow Ca2+ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca2+ oscillation may involve in the neuron-glia interaction in the striatum.The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca The endoplasmic reticular (ER) Ca2+ store is a source of [Ca2+]i elevation and is crucial for second messenger-induced intracellular Ca2+ signaling 2+ released from the ER should contribute to the modulation of neuronal signal processing in the central nervous system.The calcium ion . mGluR5 has also been suggested as a therapeutic target for Parkinson's disease 2+In the basal ganglia, the striatum receives inputs from the cortex and is thought to play a crucial role in controlling somatic motor movements, behavioral patterns, cognition, learning, and memory 2+]i oscillations in the rat striatum 2+ oscillations were not induced by action potentials, but induced by Ca2+ release from ER. In this previous study, we did not determine whether the spontaneous [Ca2+]i transients occurred in neurons or astrocytes, nor did we identify the induction mechanism of the Ca2+release from ER. Therefore, in this paper, we identified the cell types exhibiting the spontaneous [Ca2+]i transients using transgenic mice expressing green fluorescent protein (GFP) in astrocytes, and determine one of the induction mechanisms. We also analyze the cellular correlations of the slow Ca2+ oscillations. The preliminary results of this work have been previously reported in an abstract form We previously reported long-lasting spontaneous i in the slices was measured using the membrane-permeant acetoxymethyl (AM) ester of Fura-2 LR  chamber on the stage of an epifluorescent upright microscope . [Ca2+]i changes were imaged with a 20×, NA 0.95 water-immersion objective (Olympus). The Fura-2 LR-loaded slices were alternately excited at wavelengths of 340 and 380 nm using a filter changer , and fluorescent signals were captured (F340 and F380) every 2 s with a cooled CCD  or an EM-CCD . All equipment was controlled by MetaFluor software . The experiments were performed under temperature control (30±1°C).i in a fraction of the glial cells or vessel related cells, ROI was put only in the somatic region with round shape. [Ca2+]i in a striatal cell was estimated by the fluorescence ratio (R  =  F340/F380) from each imaged cell 2+]i transient for every cell was determined using an algorithm that defined the onset as the frame after which the slope of R was larger than a given set threshold. Then, the baseline was set to the mean R value of three frames before the onset, and the change in the R value from the baseline was defined as ΔR. To eliminate false positives, [Ca2+]i transients with peaks that did not exceed the threshold (ΔR  = 0.005–0.01) were discarded. Some remaining false positives were deleted upon visual inspection. When we showed the traces of the time courses of R, the baselines were subtracted from the raw R trace. The baseline of each trace was obtained by spline interpolating the local minimum values of the trace using a higher order Hanning filter  Image analysis was performed with MetaFluor and custom-made programs written in MATLAB . In the measurement of Ca2+]i transients included in the slow Ca2+ oscillations, we calculated the transient rate, amplitude, duration, rise slope, and decay slope. The transient rate was defined as the number of onsets per unit time during recording period (>1000 s). The duration indicates the time from onset until the decay to threshold. The rise and decay slopes are the slopes of a regression line for the data points in 5–90% of the peak during the rise phase and in 95% to 1/e of the peak during the decay phase of the [Ca2+]i transients, respectively.To determine the properties of individual i transients within 600 s just before the application of pharmacological agents to confirm their effects.In the pharmacological experiments, we discarded any cells that did not show [Ca2+ oscillation, we used GFAP-GFP mice. First, we confirmed that the GFP-positive cells were astrocytes and that the GFP-negative cells were neurons by immunohistochemistry . Therefore, the proportion of GFP-negative cells to S-100-positive cells was 30.3±7.3% (mean ± SD). The densities of cells positive for S-100, and for NeuN, were 636±154 , and 1538±298 , respectively . Therefore, the proportions of S-100-positive cells to the all immunostained cells were 29.2±6.4% (mean ± SD). Hence, the probability of the GFP-negative cells being astrocytes (S-100-positive cells) was 8.9±2.9%  and 95% confidence interval of this probability was 8.9±5.7%. S-100 was also expressed in the oligodendrocytes To determine whether neurons or astrocytes exhibit the slow Cahemistry . We usedhemistry . The den2+ oscillations in a field of view . Typical time courses of the slow Ca2+ oscillations are shown in 2+ transients was long (2 s)  ×10−3 Hz in astrocytes, n = 200 putative-neurons and 78 astrocytes, 15 slices, 14 mice; p = 0.0903; Mann-Whitney U test).A fluorescence image of the Fura-2 LR-loaded cells in a striatal slice of GFAP-GFP mouse is shown in 2 s) see . Therefo2+ oscillations, individual events were dissolved from the entire R trace in a record, and four parameters, the peak amplitude, duration, rise slope, and decay slope, were extracted  ×10−3, and (1.50±1.42) ×10−3 ), and those values in astrocytes were (1.32±0.77) ×10−2, 18±16 s, (1.49±1.03) ×10−3, and (1.40±1.04) ×10−3 in astrocytes , respectively. All of those four parameters were significantly different between putative-neurons and astrocytes  of the slow Ca2+ oscillations in putative-neurons as described in a later section. To determine the contribution of the Ca2+ release from the intracellular Ca2+ store to the slow Ca2+ oscillations, Ca2+ ATPases of the ER were blocked by the administration of 2 µM thapsigargin. The transient rates of the slow Ca2+ oscillations were significantly reduced by the application of thapsigargin in both putative-neurons and astrocytes , also reduced the transient rate in both types of cells (3-induced Ca2+ release from the intracellular Ca2+ store (ER) is the main origin of the slow Ca2+ oscillations.To determine the induction mechanisms of the slow Caonducted . The slo1 µM TTX , indicat3 production and is abundant in the striatum 2+ oscillations in a dose-dependent manner. When 30 µM MPEP was applied, the slow Ca2+ oscillations were almost completely blocked in astrocytes ((7.21±3.24) ×10−3 Hz for control and (0.07±0.05) ×10−3 Hz for 30 µM MPEP ; p = 0.0295 vs. control; Kruskal-Wallis test with Steel-Dwass post-hoc test), and the transient rate of the slow Ca2+ oscillations in putative-neurons was significantly reduced ((7.98±1.15) ×10−3 Hz for control and (4.16±0.97) ×10−3 Hz for 30 µM MPEP administration ; p = 0.0053 vs. control; Kruskal-Wallis test with Steel-Dwass post-hoc test). The effect of 30 µM MPEP on the slow Ca2+ oscillations in astrocytes was stronger than that in putative-neurons . Average of the transient rates of the slow Ca2+ oscillations in the condition of 10 µM MPEP administration did not change significantly compared with that in the control condition. However, 10 µM MPEP changed the some properties of the Ca2+ oscillations  ×10−2 with 10 µM MPEP administration, n = 316 events, 25 cells, 3 slices, 3 mice; mean ± SD; p = 0.0121; Kolmogorov-Smirnov test), but not in astrocytes. In the astrocytes, the decay slope was significantly decreased  ×10−4 with 10 µM MPEP administration, n = 42 events, 8 cells, 3 slices, 3 mice; mean ± SD; p = 0.0015; Kolmogorov-Smirnov test).mGluR5 is one of the receptors contributing to IPllations . The low2+ oscillations, LY367385, the specific antagonist of mGluR1, was applied. 2+ oscillations. The average values of the transient rates of the slow Ca2+ oscillations both in neurons and astrocytes did not alter with or without LY367385 significantly  ×10−3, and 1.60±1.37 ×10−3 under the control condition , and (1.25±0.77) ×10−2, 14±15 s, (1.58±0.92) ×10−3, and 1.51±0.92×10−3 with TTX administration , respectively. The distributions of the peak amplitude, duration, and rise slope of the Ca2+ transients in putative-neurons shifted toward smaller values under the TTX condition compared to the control condition  ×10−3, and (1.85±1.02) ×10−3 under the control condition  and (1.32±0.73) ×10−2, 14±9 s, (1.50±0.89) ×10−3, and (1.90±1.04) ×10−3 with TTX administration , respectively. These results indicated that blockade of action potential reduced the amplitude, the duration and the rise slope of the slow Ca2+ oscillations in only putative-neurons but not in astrocytes.To confirm the contribution of neuronal activity to the slow Ca2+ oscillations. Hence, antagonists of ionotropic glutamate receptors, 10 µM CNQX and 50 µM AP5, were applied. The distributions of those four parameters in putative-neurons and in astrocytes were shown in −2, 18±17.0 s, (1.75±1.51) ×10−3, and (1.35±1.36) ×10−3 under the control condition  and (1.56±0.90) ×10−2, 16±17 s, (1.70±1.37) ×10−3, and (1.40±1.48) ×10−3  with CNQX and AP5 administration, respectively. And, the median values of those four parameters in astrocytes were (1.64±0.71) ×10−2, 18±9 s, (1.90±1.02) ×10−3, and 1.50±1.05×10−3 under the control condition  and (1.58±0.62) ×10−2, 16±11 s, (1.70±1.05) ×10−3, and (1.60±1.19) ×10−3 with CNQX and AP5 administration , respectively. The distributions of those four parameters of the Ca2+ transients, except for a slight difference in the duration in putative-neurons  in response to activation of mGluR5 and released ATP activates adenosine or purinergic receptors in neurons 2+ signaling 2+ oscillations in neurons. The second possibility was that some kind of metabotropic receptors other than mGluR5 concerned the Ca2+ oscillations in putative-neurons. However, as described above, we could not find striking evidence for the contribution of another receptors on the slow Ca2+ oscillations in the putative-neurons. Verification of the mechanisms of the neuronal slow Ca2+ oscillations will require further investigation.The administration of MPEP almost completely blocked the slow Ca-neurons . There w2+ oscillations in both putative-neurons and astrocytes, cellular correlations of the Ca2+ elevation were reduced by the application of TTX  and large conductance (BK) channels, are expressed in the MSNs in striatum, and their currents represent between 30% and 50% of the sustained outward current 3R-dependent Ca2+ release from intracellular Ca2+ stores suppressed MSN firing via Ca2+-activated K+ channels 2+ oscillations may affect the firing rate via Ca2+-activated K+ channels 2+ oscillations reported herein may modulate the firing properties of MSNs on an intermediate time scale, one that is longer than an action potential and shorter than a circadian rhythm.Ca2+ oscillations  , duration , rise slope , and decay slope  of the Ca2+ transients in cumulative probability plots for putative-neurons and astrocytes, respectively. The solid and dashed lines indicate the distribution of each parameter for the Ca2+ transients under the control condition and during the administration of 10 µM MPEP, respectively. P- values from the Kolmogorov-Smirnov test shown in the plots. N. S.: no significant difference.(PDF)Click here for additional data file.Figure S22+ oscillations.Blocking mGluR1 did not alter the transient rate of the slow Ca A, Typical time courses of the slow Ca2+ oscillations during the administration of 50 µM LY367385 in putative-neurons and astrocytes. Horizontal bars under the time courses indicate the application period of the agents. Scale bar, 200 s, ΔR  = 0.02. B, Transient rates of the slow Ca2+ oscillations during the administration of 50 µM LY367385 in putative-neurons and astrocytes. The number of cells recorded is 26 putative-neurons and 9 astrocytes . The average values of the transient rates of the slow Ca2+ oscillations in putative-neurons were (7.59±0.95)×10−3 Hz under the control condition, and (6.94±0.76) ×10−3 Hz with LY367385 administration. The average values of the transient rates of the slow Ca2+ oscillations in astrocytes were (2.84±0.68) ×10−3 Hz under the control condition, and (3.61±0.53) ×10−3 Hz with LY367385 administration. The average values of the transient rates of the slow Ca2+ oscillations both in neurons and astrocytes did not alter with or without LY367385 significantly . N. S.: no significant difference.(PDF)Click here for additional data file.


Mrakia blollopis CBS8921T in ITS and D1/D2 sequences and had two unique characteristics when applied on an active sludge; i.e., it showed a potential to use various carbon sources and to grow under vitamin-free conditions. Indeed, it showed a biochemical oxygen demand (BOD) removal rate that was 1.25-fold higher than that of the control. We considered that the improved BOD removal rate by applying SK-4 strain was based on its lipase activity and characteristics. Finally, we purified the lipase from SK-4 and found that the enzyme was quite stable under wide ranges of temperatures and pH, even in the presence of various metal ions and organic solvents. SK-4, therefore, is a promising bio-remediation agent for cleaning up unwanted milk fat curdles from dairy milk wastewater under low temperature conditions.Milk fat curdle in sewage is one of the refractory materials for active sludge treatment under low temperature conditions. For the purpose of solving this problem by using a bio-remediation agent, we screened Antarctic yeasts and isolated SK-4 strain from algal mat of sediments of Naga-ike, a lake in Skarvsnes, East Antarctica. The yeast strain showed high nucleotide sequence homologies (>99.6%) to  Drainage from dairy parlors and milk factories produced in the process of cleaning transport pipes and milking tanks pollute rivers and groundwater with detergents, bactericides, mucus and milk fat are contaminating rivers and underground water The use of microorganisms living in polar regions for the purpose of removing nitrogen and phosphorus compounds from wastewater under low temperature conditions has been reported by Chevalier et al. Mrakia, in psychrophilic Basidiomycetous yeast, suggesting that Mrakia is a major mycoflora highly adapted to the Antarctic environment . Mrakia spp. and Mrakiella spp. are also common fungal species frequently found in cold climate areas such as Arctic, Siberia, Central Russia, the Alps and Antarctica Mrakia isolates for their ability to decompose milk fat under low temperature conditions and evaluated their potential for application to an active sludge system in a region with a cold climate. The results showed that 56 Mrakia spp. exhibited a clear zone according to fat decomposition. Antarctic yeast strain SK-4 had physiological characteristics similar to those of Mrakia blollopisIn our previous work, we examined 305 isolates of fungi including eight Ascomycetous and six Basidiomycetous species collected from Antarctica and found that they included fungi of the genus 5. We also describe identification of yeast strain SK-4 and the purification and characterization of the lipase, considered as a major enzyme to degrade milk fat in wastewater.Here we report that activated sludge containing yeast strain SK-4 has the potential to remove milk fat BODAll necessary permits were obtained for the described field studies. Permission required for field studies was obtained from the Ministry of the Environment of Japan. Sample collection in Antarctica was performed with the permission of the Ministry of the Environment of Japan.TM, BD Japan, Tokyo, Japan) at 4°C for 1 week. Yeast strain SK-4 was selectively picked for isolation on the basis of its morphology. Yeast strain SK-4 was maintained on PDA plates at 4°C and long-term storage was performed in 40% (w/v) glycerol at −80°C.Algal mat samples were collected from sediments of Naga-ike, a lake in Skarvsnes, located near Syowa station, East Antarctica. The isolate was inoculated on potato dextrose agar (PDA) (Difco-GTA ACA AGG TTT CCG T) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC). The D1/D2 domain was amplified using the following primers: NL1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG) and NL4 (5′-GGT CCG TGT TTC AAG ACG G). Sequences were obtained with an ABI prism 3100 Sequencer  using an ABI standard protocol. The ITS region and D1/D2 domain sequences of yeast strain SK-4 are deposited in DNA Data Bank of Japan (BBDJ) (Accession numbers AB630315 and AB691134). Alignment was made using CLUSTAL W  and corrected manually. Phylogenetic analysis was performed using MEGA software version 4.0 Phylogenetic analysis was done by sequencing the ITS region including 5.8S rRNA and D1/D2 domain of 26S rRNA. Cells were harvested from 2-weeks-old cultures. DNA was extracted with an ISOPLANT II kit  according to the manufacturer's protocol. Extracted DNA was amplified by PCR using KOD-plus DNA polymerase . The ITS region was amplified by using the following primers: ITS1F , 2.0 g/L of sodium nitrate , 30 g/L of carbon source and 15.0 g/L of Agar . Assimilation of nitrogen and other physiological tests were carried out according to the protocols described by Yarrow Assimilation of carbon was performed at 15°C on modified Czapek-Dox agar composed by 6.7 g/L of yeast nitrogen base without amino acids , and the other part was used as a control. Separated activated sludge was prepared with Mixed Liquor Suspended Solids  and cow's milk at 10°C with aeration. One week later, prepared activate sludge was added to cow's milk, and biochemical oxygen demand (BOD5) of waste-treated water was measured after 24 hours. BOD5 assay was carried out using a coulometer .Activated sludge (AS) was cultivated at room temperature with aeration by using cow's milk as the substrate. After one month, the sludge was divided into two parts. One part of the activated sludge was mixed with M. blollopis SK-4 was grown in YPD liquid medium  at 15°C for 96 hours at 120 rpm. After 96 hours, M. blollopis SK-4 was collected by centrifugation at 3500×g for 15 min at 4°C. The pellet was transferred to fresh cream liquid medium  and incubated at 10°C for 14 days at 90 rpm. The resulting culture was used as inoculum.2PO4, 0.29% Na2PO4, 0.02% NH4Cl, 0.04% CaCl2, 0.001% FeCl3, 0.5% yeast extract, and 1% Tween 80. The yeast was cultivated at 10°C for 324 hours at 90 rpm. One mL samples were collected every 24 h and centrifuged at 4°C for 10 min at 20000×g, and then lipase activity was measured.Lipase production medium was composed of 0.2% KH410. One unit of lipase activity was defined as the activity required to release 1 μmol of free fatty acids per minute at 30°C.Lipase activity was measured by a colorimetric method using p-nitrophenyl-palmitate as a substrate Protein concentration was measured by BCA protein assay reagent  according to the manufacturer's instructions using bovine serum albumin as a standard.M. blollopis SK-4 lipase was purified by ultrafiltration and Toyopearl-butyl 650 M  hydrophobic interaction chromatography. Four hundred mL of lipase production liquid medium was centrifuged at 4°C for 15 min at 3000×g. The supernatant was filtered through a 0.45-μm of membrane filter . The filtered medium was concentrated by ultrafiltration using an ultracel YM-30 membrane . The concentrated sample was adsorbed to a Toyopearl butyl 650 M column (2.5×20 cm) containing 1 M sodium chloride and eluted with a linear gradient from 750 mM to 100 mM sodium chloride in 20 mM Tris-HCl buffer (pH 8.5) at a flow rate of 60 mL/h. Fractions of high lipase activity was pooled and concentrated and then stored at 4°C until use. Protein molecular weight was estimated by SDS-PAGE according to Laemmli 4–C18). For determining the effects of metal ions and EDTA on lipase activity, residual lipase activity assays were carried out under standard assay conditions with final concentrations of 1 mM of various bivalent metal ions and EDTA. Lipase activity assay in the absence of metal ions and EDTA was carried out as a control. Optimum pH was measured at 30°C for 30 min and determined at various pH values of 50 mM buffer as follows: sodium citrate (pH 3.0–5.0), sodium phosphate , Tris-HCl (pH 7.5 and 8.5), glycine-NaOH (pH 9.0) and sodium carbonate .Substrate specificity was determined by using substrate as different p-nitrophenyl esters (COptimum temperature was measured in 50 mM sodium phosphate buffer (pH 7.0) for 30 min. To determine the pH stability of lipase, the enzyme was preincubated in various buffers for 15 h at 30°C and then adjusted to pH 8.5. The residual enzyme activity was measured by using p-nitrophenyl-palmitate as a substrate at 65°C for 30 min. For determining thermo-stability, lipase was preincubated for 30 min at different temperatures and the residual activity was measured at 65°C for 30 min in 50 mM Tris-HCl (pH 8.5). Effects of organic solvents on lipase activity were determined at 65°C for 30 min in 50 mM Tris-HCl (pH 8.5) containing various organic solvents at final concentration of 5% (v/v). Lipase activity assay in the absence of organic solvents was carried out as a control.Mrakia blollopis CBS 8921T . Activated sludge containing l 63.8%, . When BOl 65.2%, . RegardlM. blollopis SK-4 also produced the enzyme. It is known that the production of lipase from Candida rugosa increased when yeast extract was used as a nitrogen source M. blollopis SK-4 were obtained. When 0.5% (w/v) yeast extract was used as a nitrogen source in the medium, lipase production by M. blollopis SK-4 lipase increased dramatically after 180 h. Maximum lipase activity of 0.695 U/mL was obtained with 0.5% (w/v) yeast extract, as compared to 0.059U/mL in its absence, after 324 h of inoculation, after which the accumulation of lipase markedly decreased .Many microorganisms are known to produce lipase using Tween 80 as a substrate M. blollopis SK-4 morphology was observed by the fluorescence in situ hybridization (FISH) method during secretion of lipase. Therefore, all of the morphology of M. blollopis SK-4 during secretion of lipase was yeast form (data not shown).Lipase production medium was centrifuged at 3000×for 15 min at 4°C. The supernatant was filtered through a 0.45-μm membrane filter. The filtered solution was concentrated by ultrafiltration. After ultrafiltration, 72.3% of total lipase activities were recovered and 2.2-fold lipase specific activity was obtained. Then, enzyme solution was applied on a Toyopearl butyl-650 M column (2.5×20 cm) and purified by single-step hydrophobic interaction chromatography. Finally, 9.4% of the enzyme was recovered and 20.1-fold of specific activity, compared to crude sample, was obtained with a specific activity of 51.7 U/mg . The purcat  = 3.93 and 4.04 S−1). At temperatures of 80°C and 95°C, 30.5% (kcat  = 1.23 S−1) and 6.8% (kcat  = 0.27 S−1) of enzyme activity were retained (cat  = 5.12 S−1) of residual enzyme activity even after 30-min preincubation. At 75°C, 80°C and 85°C, 48.2% (kcat  = 2.51 S−1), 41.8% (kcat  = 2.18 S−1) and 28.03% (kcat  = 1.46 S−1) of enzyme activity remained after 30-min preincubation (−1), whereas 57.0% (kcat  = 0.86 S−1), 63.5% (kcat  = 0.96 S−1) and 27.8% (kcat  = 0.42 S−1) of the enzyme activity was retained at pH 7.0, pH 9.3 and pH 9.7 compared to 100% at pH 8.5 (cat  = 2.38 S−1) of the enzyme activity remained at pH 3.0 even after preincubation for 15 h at 30°C . The substrate specificity was determined in the presence of 1 mM p-nitrophenyl esters as a substrate with 50 mM sodium phosphate (pH 7.0) at 30°C for 30 min. Relative activity toward C4−C14 was more than 100% compared to 100% toward p-nitrophenyl-palmitate. C18 had relative activity of 71.6%  enhanced SK-4 lipase acidity. Solvents such as acetone and chloroform however, had only a slight inhibitory effect in the activity, with relative activities of 77.9% and 70.4%, respectively  and lipase production medium without yeast extract (•).(TIF)Click here for additional data file.Figure S2SDS-PAGE of purified lipase from Mrakia blollopis SK-4. Lane 1. Molecular weight marker; 2. Purified lipase.(TIF)Click here for additional data file.


To achieve protection by silibinin, the drug was applied to the cell cultures for 2 h in advance. OTA stimulated lipid peroxidation on cultured immortalized rat liver HPCT cells, as was revealed by malondialdehyde (MDA) production. Lipid peroxidation occurred further by H2O2 and ActD/TNF-α incubation. These reactions were also suppressed by silibinin pretreatment. We conclude that the anti-apoptotic activity of silibinin against OTA, H2O2 and ActD/ TNF-α is caused in vitro by the antioxidative effects of the flavanolignan. Furthermore, cytotoxicity of the pro-apoptotic toxins was revealed by MTT-test. When applied separately, ActD and TNF-α showed no cytotoxic effects after 24 h, but were cytotoxic if applied in combination. The used concentrations of OTA, H2O2 and the dose of UV-C caused a substantial decrease in viability within 36 h that was prevented mostly by silibinin. We conclude that silibinin is a potent protective compound against apoptosis and cytotoxicity caused by OTA and the investigated compounds. In cultures of primary rat hepatocytes, apoptosis occurred after application of 20 ng/mL tumor necrosis factor alpha (TNF-α). However, this was only in the presence of 200 ng/mL of the transcriptional inhibitor actinomycin D (ActD). This toxic effect was completely prevented in the presence of 25 µg/mL soluble TNF-α receptor I (sTNFR I) in the supernatant of hepatocyte cell cultures. Apoptosis also occurred after application of 12.5 µmol/L ochratoxin A (OTA). However, that was not prevented by up to 500 µg/mL sTNFR I, indicating that TNF-α/TNFR I is not involved in OTA mediated apoptosis in hepatocytes. The antioxidative flavanolignan silibinin in doses from 130 to 260 µmol/L prevented chromatin condensation, caspase-3 activation, and apoptotic DNA fragmentation that were induced by OTA, by 10 mmol/L hydrogen peroxide (H This program permits the removal of damaged cells in multicellular tissue forming-organisms [Liver injury by hepatotoxins may merge in liver cell apoptosis, rganisms . It resurganisms ,3.in vivo by apoptotic changes in rat and mouse livers [in vitro [in vitro, such as ActD [2O2 and UV-C light in the absence and presence of the membrane protecting antioxidant silibinin. We show that OTA-mediated apoptosis is likely to be caused by oxidative stress, and that silibinin blocked apoptosis and cytotoxicity. That flavanolignan is extracted from the milk thistle Silybum marianum and was used here as a potent antioxidant [OTA is an unavoidable mycotoxin contaminant of feed and foodstuff that mainly causes nephrotoxicity, as well as hepatotoxicity ,5. The he livers ,7, but ain vitro ,9. The ain vitro  that is in vitro . The maiin vitro ,12,13,14 as ActD ,16. Howeioxidant ,18,19 anioxidant . Silibinioxidant . Liver toxicity is one of the consequences of OTA, but the mechanisms by which OTA exerts its hepatotoxicity are still scantily understood. One possibility is oxidative stress of liver cells by OTA via lipid peroxidation, since the release of ROS under OTA was reported from rat liver ,23. AnotOTA is hepatotoxic in rats, perhaps as a consequence of oxidative stress . It was OTA is cytotoxic on cultured primary rat hepatocytes in a dose- and time-dependent manner a. At 2.5Cell death by TNF-α in cultured hepatocytes becomes manifested only under the metabolic condition of the transcriptional arrest . Therefo2O2 [2O2 and UV-C resulted in a dose-and time-dependent cytotoxicity  and paracetamol (AAP) through counteracting lipid peroxidation and enzyme leakage [2O2 or with 50 mJ/cm2 UV-C. Under all these conditions, pretreatment for 2 h with silibinin at 130 µmol/L protected the cells as confirmed by MTT-test. Under the same experimental conditions, even the ActD/TNF-α-mediated hepatotoxicity was blocked by silibinin albeit at a higher concentration of 260 µmol/L  prevented caspase-3 activation by ActD/TNF-α. Others also found that silibinin inhibits caspases in various cell systems and conditions: it completely protects ECV-304 cells against H2O2 induced injury mediated by caspase-3 [In our case, the exposure of hepatocyte cultures to OTA, ActD/TNF-α, Hotection . As showaspase-3 , reverseaspase-3 , alleviaaspase-3 , preventaspase-3  and in caspase-3 . 2O2 and UV-C. Treatment of primary rat hepatocytes by OTA, ActD/TNF-α, H2O2 and UV-C led to a statistically significant induction rate of nuclear apoptotic events on approximately 25, 62, 55 and 30% of cells, respectively  triggers apoptosis by binding TNF-α with its extracellular domain and by activation of its intracellular domain, called adaptor protein TNFRI associated death domain (TRADD). Following activation of TRADD, the recruitment and activation of the initiator aspartate specific cysteine protease caspase-8 occurs, which triggers further events of receptor induced apoptosis ,50. In o2O2. The results indicated that OTA generates, as with the other hepatotoxins, oxygen radicals in cell cultures  ,52 were cultures .MW 482.44 g/mol), Recombinant Rat Tumor Necrosis Factor-α (TNF-α), fetal bovine serum, dexamethasone, inosine, trypan blue and Hoechst stain 33342 were purchased from Sigma Aldrich, Steinheim, Germany. Penicillin, streptomycin, trypsin/EDTA, sodium pyruvate and Dulbeccos modified Eagles medium (DMEM) low glucose were purchased from PAA Laboratories, Cölbe, Germany. Ochratoxin A (MW 403.81 g/mol) was purchased from CSIR Food Science and Technology, Pretoria, South Africa. Hydrogen peroxide (H2O2), DMSO, agarose NEEO Ultra-Quality, and MTT -2,5-diphenyl tetrazolium bromide) were purchased from Roth, Karlsruhe, Germany. HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and collagen R and recombinant human insulin were purchased from SERVA Electrophoresis GmbH, Heidelberg, Germany. Proteinase K, RNase and DNA ladder markers were purchased from Fermentas, St. Leon-Rot, Germany. Soluble tumor necrosis factor receptor I (sTNFRI) was kindly obtained from Prof. Dr. Joachim Roth, Giessen.Silibinin . The animals were fed in situ perfusion method as described in [2 in air. The culture medium was replaced, and the compounds were applied. Control cells were treated with equal volume of DMSO as a vehicle for OTA and silibinin. Final DMSO concentration was 0.5% for all treatments.Male Wistar rats were anesthetized with urethane 20% . Hepatocytes were prepared by EDTA ribed in . The celAfter replacement of the culture medium, the isolated, adherent rat hepatocytes were pre-incubated with ActD (200 ng/mL) for 30 min, and then rat recombinant TNF-α was added to some dishes at a concentration of 20 ng/mL, unless otherwise indicated.in vitro model has to be proven in order to replace in vivo experiments [v/v) fetal calf serum, 2 mM L-glutamine, 10 µg/mL insulin, 10 µg/mL inosine, 1.5 µmol/L dexamethasone, 100 IU/mL penicillin and 100 mg/mL streptomycin [Because HPCT-1E3 cells are a good model for studying cytotoxicity and express several hepatocyte specific properties in contrast to other immortal cell lines, their suitability as an eriments . The ratptomycin .2O2 and UV was determined by the 3--2,5-diphenyl tetrazolium bromide (MTT) assay [4 cells/0.1 mL DMEM were seeded in 96-well collagen-coated tissue culture plates. After 3 h incubation, the cells were treated with different concentrations of either ActD at 0, 50, 100, 200 and 333 ng/mL, or with TNF-α at 0, 10, 15, 20 and 30 ng/mL. Other cells were pre-incubated with 200 ng/mL ActD 30 min, prior to TNF-α, at the given concentrations for 24 h; others were treated with 0, 0.5, 2.5, 12.5 µmol/L OTA, 0, 5, 10 and 15 mmol/L H2O2 or irradiated with UV light  at 0, 25 and 50 mJ/cm2, and all were incubated for 24 and 36 h. To assess the effect of silibinin on the cytotoxicity, cells were pre-treated with and without silibinin at the given concentrations 2 h prior to the administration of ActD/ TNF-α at 200/20 ng/mL or 12.5 µmol/L OTA or 10 mmol/L H2O2, or irradiation with UV light at 50 mJ/cm2. After that, the cells were incubated with 10 µL of MTT solution (5 mg/mL) for 3 h. The formation of formazan crystals were tracked occasionally under the microscope. Subsequently, the medium was aspirated and the formazan crystals were solubilized by adding 100 µL isopropanol solution (98.86 mL 2-propanol and 1.14 mL HCl) and mixed to ensure the dissolving of crystals. Finally, the absorbance of each well was measured by a Benchmark microplate reader at dual wavelength (550 nm and 655 nm). Within 1 h of adding the isopropanol, plates were read. Cell viability was expressed as percentage of formazan absorbance of non-treated viable cells under each treatment regimen.The concentration dependency of cytotoxicity of ActD, TNF-α, HT) assay , detecti2O2 and UV, we stained the chromatin of fixed primary hepatocyte monolayers with DNA binding fluorochrome Hoechst stain 33342 and counted apoptotic nuclei using fluorescence microscopy. Briefly, 1 mL cell suspension containing 2 × 105 cells were seeded on collagen-coated 2 well Lab-tek tissue culture chamber/slides. After 3 h of incubation at 37 °C, the cells were pre-treated with and without silibinin at the given concentrations 2 h prior to the administration of 12.5 µmol/L OTA, ActD/TNF-α 200/20 ng/mL separately and in combination, 10 mmol/L H2O2 or irradiated with UV light  at a dose of 50 mJ/cm2 for 24 h. After that, the cells were washed with cold PBS once and fixed with 2% paraformaldehyde for 10 min. Then, Hoechst 33342 solution was added at 1 µg/mL for 5 min at room temperature in darkness. Finally, stained cells were washed with PBS again, mounted and visualized under a fluorescence microscope. Fluorescence imaging was performed on Nikon Eclipse 80i fluorescence microscope supplied with DAPI filter  from Japan. The images were captured with the NIS elements software (V 3.10). Finally, the apoptotic nuclei were counted using Adobe Photoshop CS5 extended version 12.1. The percentage of apoptotic nuclei is given as apoptotic index.In order to visually assess apoptosis induction by OTA, ActD/TNF-α, H2O2 or irradiation with UV light at a dose of 50 mJ/cm2 and at 260 µmol/L 2 h prior to ActD/TNF-α 200/20 ng/mL for 12 h. The cells were harvested after 12 h without trypsinization by a cell scraper from the incubation medium and centrifuged at 420× g for 5 min at 25°C. The final pellet was re-suspended in 50 µL of chilled Cell Lysis Buffer (PromoCell) and incubated on ice for 10 minutes followed by centrifugation at 13,000× g for 1 min. The supernatant (cytosolic extract) was transferred to a fresh tube and immediately stored at −80 °C. The protein content of the supernatant was reliably estimated with BCA protein assay kit (Novagen). For colorimetric measurement of caspase-3 activity, 150 µg protein of the cytosolic extract was diluted to 50 µL Cell Lysis Buffer for each sample. Subsequently, 50 µL of 2× Reaction Buffer containing 10 mmol/L DTT and 5 µL of the 4 mmol/L DEVD-pNA substrate was added and then incubated at 37 °C for 2 h. The enzyme activity (color intensity from DEVD-pNA cleavage) was measured at 405 nm with a microplate reader.Caspase-3 activity was determined using Caspase-3/CPP32 Activity Colorimetric Assay Kit according to the company instructions . The assay is based on the spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA. Briefly, isolated primary rat hepatocytes were seeded in 94 mm Petri dishes and incubated for 3 h. Then, the cells were pre-treated with and without silibinin at 130 µmol/L 2 h prior to the administration of 12.5 µmol/L OTA or 10 mmol/L H2O2 or irradiation with UV light or at 260 µmol/L 2 h prior to ActD/TNF-α for 24 h. Other cells were pre-treated with sTNFRI 2 h prior to the administration of OTA and ActD/TNF-α. Cells were harvested after 24 h as described above in the incubation medium and were spun down at 500× g for 5 min at 25 °C, re-suspended in PBS and spun down again. Genomic DNA was isolated according to Wörner and Schrenk [Isolated primary rat hepatocytes were seeded in 94 mm Petri dishes and incubated for 3 h. Then the cells were pre-treated with and without silibinin at 130 µmol/L 2 h prior to the administration of OTA, H Schrenk . DNA conThe oxidative stress was assessed: (1) fluorometrically by measuring malondialdehyde (MDA) as an end product of lipid peroxidation using Abcam’s Lipid Peroxidation Assay Kit  and (2) colorimetrically by determining ROS generation using ROS ELISA kit . 6 cells) on 35 mm Petri dishes and pre-incubated overnight. Then, the cells were pre-treated with and without silibinin at 260 µmol/L 2 h prior to the administration of 50 µmol/L OTA, ActD/TNF-α 200/20 ng/mL and 10 mmol/L H2O2 for 6 h. The cells were harvested after 6 h without trypsinization by a cell scraper from the incubation medium and centrifuged at 3000 rpm for 3 min at 25 °C. The final pellet was used for measuring MDA and ROS according to the company’s instructions.HPCT-1E3 cells were seeded at .in vitro cytotoxicity and apoptosis in rat hepatocyte cell cultures that were independent on TNF-α and were, thus, different from in vivo conditions in the intact rat liver. OTA lacked binding to soluble TNF-α receptor I (sTNFRI). In cell cultures, toxicity of OTA and ActD/TNF-α was conveyed by oxidative stress and was comparable to H2O2. The herbal flavanolignan silibinin was a potent cytoprotective and anti-apoptotic agent that counteracted the cytotoxicity and induction of apoptosis by OTA, ActD/TNF-α, H2O2 and UV-C on primary rat hepatocytes cultures. The powerful hepatoprotective effects of silibinin were observed in a dose-dependent manner when the compound was added in advance. This distinguishes silibinin as a prophylactic hepatoprotective remedy. The mycotoxin ochratoxin A caused


TNF-α, cytosolic Cytochrome C were seen in bothHCT15 cells HT29 treated with plumbagin, aberrant apoptosis with decreased levelof pEGFR, pAkt, pGsk-3β, PCNA and Cyclin D1was observed only in 15 µMand 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29cells. This suggests that plumbagin induces apoptosis in both HCT15 cells andHT29 treated, whereas, proliferation was inhibited only in 15 µM and 30µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells,but not in 50 µM plumbagin treated HT29 cells. Expression of COX-2 wasdecreased in 75 µM plumbagin treated HT29 cells when compared to 50µM plumbagin treated HT29 cells, whereas HCT15 cells lack COX. Hence theobserved resistance to induction of apoptosis in 50 µM plumbagin treatedHT29 cells are attributed to the expression of COX-2. In conclusion, plumbagininduces apoptosis in colonic cancer cells through TNF-α mediated pathwaydepending on expression of COX-2 expression.Plumbagin, a quinonoid found in the plants of the Plumbaginaceae, possessesmedicinal properties. In this study we investigated the anti-proliferative andapoptotic activity of plumbagin by using two human colonic cancer cell lines,HT29 and HCT15. IC50 of Plumbagin for HCT15 and HT29 cells  were significantly different. To study the responseof cancer cells during treatment strategies, cells were treated with twodifferent concentrations, 15 µM, 30 µM for HCT15 and 50 µM, 75µM for HT29 cells. Though activation of NFκB, Caspases-3, elevatedlevels of  Colorectal cancer (CRC) is a primary public health concern to the humankind and isthe third most common cancer in the United States NF-κB is a key inflammatory mediator involved in initiation, progression andmetastasis of CRC Plumbagozeylanica L. . Plumbagin has beenshown to exert anticarcinogenic, antiatherosclerotic and antimicrobial effects P. zeylanica L. has been used in Indian medicine for ∼2,750years and its component possess antiatherogenic, cardiotonic, hepatoprotective andneuroprotective properties Plumbagin , a quinonoid, found in the plantsof the Plumbaginaceae, Droseraceae, Ancestrocladaceae, and Dioncophyllaceaefamilies. The Chief source of Plumbagin is the root of in vitroexperimental models using two human colonic cancer cell lines, HT29 and HCT15.Further, the mechanism of anticancer activity of plumbagin was established byanalysing cell cycle regulation and signalling molecules related to cell survivaland apoptosis.Sugie et al. 2, tissue culture flasks  at 37°Cunder 5% CO2 and 95% air.Human colon tumor cell lines HT29 and HCT15 were obtained from NCCS Pune. Cellswere grown in Dulbeccos Modified Eagle Medium supplemented with 10% FBS , 100 units/ml Penicillin, 100µg/ml Streptomycin, 10–20 µg/ml fungisone , pH7.4 in 25 cmPlumbagin was purchased from Sigma. A 100 mM solution of plumbagin was preparedin dimethyl sulfoxide (DMSO), stored as small aliquots at −20°C andthen diluted as needed in cell culture medium. Dose-response studies werecarried out to determine the suitable dose for the inhibition of cell growth andinduction of apoptosis.3 per well) were seededin a flat-bottomed 96-well plate and incubated for 24 h at 37°C and in5% CO2. Both cell lines were exposed to plumbagin . The solvent DMSO treated cells served as control. Cells were thentreated with MTT reagent (10 µl/well) for 4 h at 37°C and thenisopropanol (100 µl) was added to each well to dissolve the formazancrystals. The optical density (OD) was recorded at 570 nm in a microplatereader. Percentage of residual cell viability was determined as[1−(OD of treated cells/OD of control cells)]×100.Sensitivity of HT29 and HCT15 cells to plumbagin was determined by the MTTcolorimetric assay PBMC was isolated from heparinized venous blood obtained from a healthy humanvolunteer by Ficoll-Paque  densitygradient centrifugation as per standard procedure 5 cells per well. Cells were trypsinized and harvestedby centrifugation at 1700 g. Flow cytometric analysis of cellcycle was performed by staining of permeabilized cells with propidium iodide(PI) for DNA content. Cells were resuspended in PBS, fixed with 70%ethanol, stained with PI solution  and incubated for 30 min at room temperature (RT) indarkness. Fluorescence intensity was measured by flow cytometry(Becton-Dickinso) using excitation and emission wavelengths of 488 and 525 nm,respectively. All experiments were performed in triplicates.Both HT29 and HCT15 cells were seeded in a 6-well plate at a density of1×105) were seededinto 24-well plates and exposed to the desired concentrations of plumbagin  for specified time periods.Following treatment, cells were pelleted by centrifugation at 1500 rpm. Thepellet was resuspended in 60 µl of PBS (pH 7.4). 10 µl of cellsuspension was mixed with 100 µl of 1% low melting agarose and 75µl of this cell–agarose mixture was spread on microscopic slidesprecoated with 1% agarose. A third layer of 0.5% low meltingagarose (75 µl) was applied over the layer of agarose with the cellsuspension. Slides were incubated for 1 h in a lysis solution .Subsequently cells were exposed to an alkaline buffer  for 30 min. The microscopic slides were subjected toelectrophoresis at 0.8 V/cm−1 for 30 min after which slides were immersedin a neutralization buffer  for 15 min. After staining withethidium bromide (20 µg/ml) slides were analyzed under a fluorescencemicroscope (Nikon PCM-2000). Images of at least 50 cells from three slides wereanalyzed using Cometscore™ software. For each comet two areas wereselected: the whole cellular DNA and an area containing only the head region ofthe comet. In each selection the densities were measured and the results werepresented as tail moment defined as the result of the percentage of DNA in thetail multiplied by the tail length.The comet assay was performed according to the method of Singh et al. (1988)7 cells, 1 ml ofTri reagent was added and lysed by repetitive pipetting and allowed to stand for5 min followed by addition of 200 µl of chloroform for phase separation.Vigorously vortexed for 15 sec and allowed to stand for 15 min followed bycentrifugation at 12000 g for 15 min at 4°C. The upper aqueous layercontaining RNA was transferred to a fresh sterile DEPC treated microfuge tube.To this, 500 µl of ice cold isopropanol was added, gently mixed andallowed to stand for 10 min and centrifuged at 12000 g for 15 min at 4°C.The supernatant was discarded and the RNA pellet was washed with 1 ml of75% ethanol in DEPC treated water and again centrifuged at 14000 g for 10min at 4°C to get total RNA. This pellet was dissolved in 25 µl ofsterile RNase free water by heating at 55°C for 20 min and stored at−20°C until use.For preparation of total RNA, the phenol – guanidinium thiocyanate basedTri Reagent  was used. To 10To synthesize cDNA, a reverse transcription reaction solution containing 1.0µg total RNA in RNase/DNase-free water and 1.5 µl of random hexamerprimer  were incubated for 10 min at 72°C and chilledimmediately. To this, 5.0 µl premixed 10 mM dNTP solution , 3.0 µl 10× M-MLV reverse transcriptase buffer , 1.0 µl (200 units/µl) M-MLV reverse transcriptase, were added and made up to 50 µl using sterileRNase/DNase-free water.http://fokker.wi.mit.edu/primer3/input.htm and nucleic acidsequence was accessed from http://www.ncbi.nlm.nih.gov/entrez/viewer.fci?val=Reference IDTable. The primers were purchased from Integrated DNA technologies, USA.Further, expression of COX-2 and TNF-α was analysed semi quantitativelyusing UN SCAN IT software.To amplify the cDNA, polymerase chain reaction (PCR) ready mix  was used according to manufacturer's instruction. All PCRsamples were denatured at 94°C for 5 min prior to cycling and were extendedfor 10 min at 72°C following cycling. The PCR assay using primers wasperformed for 39 cycles at 94°C for 60 s, 60°C for 60 s, and 72°Cfor 60 s. Primers for GAPDH, COX-2, TNF-α are shown in To 0.5 ml phenol-ethanol supernatant, 3 volumes of acetone were added toprecipitate proteins. Samples were mixed by inversion for 10–15 sec toobtain a homogeneous solution and allowed to stand for 10 min at roomtemperature. Protein was precipitated by centrifuging at 12,000 g for 10 min at4°C. The supernatant was removed and pellet dispersed in 0.5 ml of 0.3 Mguanidine hydrochloride in 95% ethanol with 2.5% glycerol(V∶V). After dispersing the pellet, another 0.5 ml aliquot of theguanidine hydrochloride/ethanol/glycerol wash solution was added to the sampleand stored for 10 min at room temperature. Protein was pelleted at 8,000 g for 5min at 4°C. The wash solution was removed and two more washes were performedin 1 ml each of the guanidine/ethanol/glycerol wash solution. Final wash wasperformed with 1 ml of ethanol containing 2.5% glycerol (V∶V).Protein was pelleted out by centrifuging at 8000 g for 10 min at 4°C and airdried for 5 min. After briefly air-drying, the protein pellet was dissolved in1% SDS. Protein concentrations of cell lysates were estimated accordingto Lowry et al., 1951 5 cells per well were seeded in 2 ml antibiotic-freenormal growth medium supplemented with FBS. Cells were incubated at 37°C ina CO2 incubator until the cells were 60–80% confluent.Cell viability was assessed before transfection. The following solutions wereprepared: Solution A: For each transfection, diluted 6 µl of siRNA duplex into 100 µl siRNATransfection Medium . Solution B: For each transfection, dilute6 µl of siRNA Transfection Reagent  into 100 µlsiRNA Transfection Medium. siRNA duplex solution (Solution A) was mixed withdiluted Transfection Reagent (Solution B) and incubate the mixture 45 minutes atroom temperature. For each transfection, 0.8 ml siRNA Transfection Medium wasadded to each tube containing the siRNA Transfection Reagent mixture (Solution A+ Solution B), mixed gently, overlaid onto washed cells and Incubated for5–7 hours at 37°C in a CO2 incubator. After incubation, 1ml of normal growth medium containing 2 times the normal serum and antibioticsconcentration  were added without removing thetransfection mixture. Cells were incubated for an additional 18–24 hours.Medium was replaced with 1 ml of fresh 1× normal growth medium and usedfor further assay.COX-2 gene was silenced by transfecting COX-2 siRNA. In a six well tissue cultureplate, 2×10Values were recorded as the mean x ± SD of three experiments. Experimentalresults were analyzed by student's t-test. P<0.00 was considered as thelevel of highly significance and P<0.05 was considered as significance forvalues obtained for treated groups compared with control group.50 at 24 hours was 22.5 µM, whichis significantly lower than that of IC50 of plumbagin treated HT29,which is 62.5 µM. Only non significant changes in viability of PBMC's were observed even in100 µM of Plumbagin incubated cells and cytosolic release of cytochrome Cwere analysed by western blotting . COX-2 siRNA transfected HT29 cells treated with 50 µM ofplumbagin showed 36.78% of cells in GRelative expression of COX-2 in Plumbagin treated COX-2 si RNA transfected HT29cells are significantly (P<0.001) decreased when compared to scrambled si RNAtransfected HT29 cells treated with Plumbagin (2) was unchanged in Plumbagin treatedHCT15 as compared to that of the control HT15 cells. Significantly (P<0.001)decreased level of PGE2 was observed in Plumbagin treated COX-2 siRNA transfected HT29 cells when compared with Plumbagin treated HT29 cells andscrambled si RNA transfected HT29 cells treated with Plumbagin , known as prostaglandin (PG) H2 synthase, is the rate-limitingenzyme in the conversion of arachidonic acid into PGs. Overexpression of COX2 hasbeen frequently observed in colon tumors and COX2 plays a major role in coloncarcinogenesis 50 value of HT29 cells (62.5 µM) was much higherthan that of HCT cells (22.5 µM). Hence, further study was performed bytreating cells with two different concentrations, one concentration fixed well belowthe IC50 value  while the other concentration was fixed above theIC50 . This has been done to study the response of cancer cells duringtreatment strategies. However, plumbagin did not exhibit any significant toxicity onnormal cells (PBMC) at any of the above concentrations, suggesting that plumbaginpossesses selectivity between normal and cancer cells.In our study, we demonstrate that in a dose-dependent manner plumbagin inducescytotoxicity effectively in HCT15 cells when compared to the HT29 cells.Accordingly, IC50. However, treatmentof 75 µM concentration of Plumbagin induced apoptosis in HT29 cells.Proliferation of HCT15 cells was inhibited at both 15 µM and 30 µMconcentration of plumbagin, whereas, Proliferation was inhibited only in 75 µMplumbagin treated HT29, while, 50 µM plumbagin treated HT29 cells tend toproliferate. PCNA data also confirmed the above fact. The above fact is attributedto the observed resistance to induction of apoptosis by Plumbagin in HT29 cells whentreated at 50 µM plumbagin.Plumbagin alone has been shown to induce apoptosis in different cell types. Our cometassay data also suggest that both the concentrations of Plumbagin induce apoptosisin HCT15. Interestingly, HT29 cells showed resistant to Plumbagin induced apoptosisat 50 µM concentration which is below the ICPrevious reports have suggested that plumbagin induce apoptosis through theactivation of caspases-3 and Cytochrome-C cox-2 transcripts in 75 µM plumbagin treated HT29indicates that COX-2 plays a crucial role in plumbagin induced apoptosis. Further,lack of expression of cox-2 transcripts in control and plumbagintreated HCT15 authenticates the above facts. However, only non significant changeswere observed in the level of NFκB transcripts.Since the only difference between these two cell lines are COX-2 and that couldmediate a balance between apoptosis and cell survival, we analysed the expression ofCOX-2 along with its transcription factor NFκB. Decreased expression ofAs COX-2 is regulated by activated NFκB, we analysed activation NFκB byimmuno blotting with p65 specific antibodies. Our results indicate that NFκBactivation was found elevated in Plumbagin treated HCT15 and HT29 cells at both theconcentrations. Activation of NFκB (p65) is due to elevated levels of TNF-αthrough TRADD-TRAF. Expression of COX-2 is decreased even at high levels of p65(activated NFκB) which cloud be due to the fact that plumbagin modulates p65 bytargeting Cystine-38. Modification of Cystine-38 in p65 affects its DNA bindingcapacity Studies in human cancer indicate that use of specific COX2 inhibitors may be aneffective approach for colorectal cancer prevention and treatment 0/G1 phase due to degradation of cyclin D1 by activeGsk-3β as Akt in its inactive form may be due to unavailability of COX-2dependent PGE2. Akt is still active due to COX-2 dependent phosphorylation of EGFRthrough PGE2 in 50 µM plumbagin treated HT29 cells showing accumulation ofcyclin D1 causing cell cycle disregulation and forms the basis for cellproliferation even with 50 µM plumbagin. Further, increased apoptosis Sub-G1population and increased cytotoxicity associated with decrease levels of PGE2 in 50µM Plumbagin treated COX-2 si RNA transfected HT29 cells, authenticate thatthe COX-2 plays crucial role in resisting Plumbagin induced apoptosis of HT29cellsActivation of Akt inhibits Gsk-3β and causing accumulation of cyclin D1 leadingto loss of cell cycle regulation Taken into all above facts, we conclude that induction of apoptosis in colonic cancercells by plumbagin is mediates through TNF-α expression and TNF-α mediatedpathway by activating Caspase-3 and releasing of Cytochrome C. However, balancebetween cell survival and apoptosis controlled by COX-2. Modulation of p65(NFκB) by Plumbagin inhibits cell survival through inhibiting of phosphorylationof EGFR, Akt and GSK-3β and shifts the balance towards apoptosis.


In vitro. We exposed A549, H1650 and CNE-2Z cell lines to metal oxide NPs, and found CuO NPs to be the most toxic, SiO2 mild toxic, while the other metal oxide NPs had little effect on cell viability. Furthermore, the autophagic biomarker LC3-II significantly increased in A549 cells treated with CuO NPs, and the use of the autophagy inhibitors wortmannin and 3-methyladenin significantly improved cell survival. These results indicate that the cytoxicity of CuO NPs may involve the autophagic pathway in A549 cells.Metal oxide nanoparticles (NPs) are among the most highly produced nanomaterials, and have many diverse functions in catalysis, environmental remediation, as sensors, and in the production of personal care products. In this study, the toxicity of several widely used metal oxide NPs such as copper oxide, silica, titanium oxide and ferric oxide NPs, were evaluated  Metal oxide nanoparticles have been used to produce a wide range of products. Their industrial and commercial applications include catalysis, sensors, environmental remediation, personal care products, cosmetics, and they are showing great prospect in the field of medicine, including imaging and drug delivery 2, TiO2, Fe2O3 and Fe3O4. Furthermore, the mechanism of cell death caused by metal oxides NPs was investigated.Nanoparticles have a small aerodynamic diameter. They can easily penetrate lung tissue and cause adverse pulmonary reactions Copper (II) oxide (<50 nm), iron (III) oxide (<50 nm), iron  oxide (<50 nm), and silicon dioxide (5–15 nm) nanopowder were purchased from Sigma Aldrich; titanium (IV) oxide anatasenanopowders (10 nm and 32 nm) were purchased from Alfa Aesar. 3-Methyladenine, wortmannin, zVAD-fmk, necrostatin 1, rapamycin and bafilomycin A1 were purchased from Sigma. siRNAs against human Atg5 (5′-ACCGGAAACUCAΜGGAAUAdTdT-3′/3′-dTdTΜGGCCUUΜGAGUACCUUAU-5′) were purchased from RiBo Biotechnology. Transfection reagent X-treme Gene HP was purchased from Roche and Lipo2000 was purchased from Invitrogen.LC3B and cleaved caspase-3 primary antibodies were purchased from Cell Signaling Technologies. Anti-β-actin antibody was purchased from Sigma-Aldrich. Horseradish peroxidase-conjugated secondary antibodies and western blot luminal reagents were purchased from Santa Cruz Biotechnology. The Celltiter 96 Aqueous One Solution Cell Proliferation Assay kit was obtained from Promega. The in situ cell death detection kit-POD was purchased from Roche.The EGFP-LC3 plasmid, which encodes a fusion protein of enhanced green fluorescent protein (EGFP) and LC3, was constructed by K.Kirkegaard  and was obtained from ADDGENE.2. NCI-H1650 and CNE-2Z were culturedin RMPI-1640 medium at 37°C and 5% CO2. Nanoparticles were suspended in culture medium at a concentration of 1 mg/ml, and then sonicated in a sonicator bath for 30 min. The solution was then diluted with medium to a concentration of 30 µg/ml. The dilutions of NPs were vigorously vortexed for 30 s prior to cell exposure to avoid nanoparticle agglomeration. Bafilomycin was diluted in DMSO at a concentration of 50 µM and then add to the cells at a concentration of 50 nM 1 h before exposure to nanoparticles.Human adenocarcinoma A549 cells, human non-small cell lung cancer H1650 cells and human nasopharyngeal carcinoma CNE-2Z cells were provided by American Type Culture Collection (ATCC). The human type II alveolar epithelial cell line (A549) was cultured in F12/HAM’s (Hyclone) medium supplemented with 10% FBS and 100 U/mlpenicillin-streptomycin at 37°C and 5% CO5/ml in96-well plates and then exposed to metal oxide NPs 12 h later at a concentration of 30 µg/ml for 24 h, the function of bafilimycin A1 on A549 cells were treated with CuO nanoparticles of 30 µg/ml for 18 h. For each well, the medium was removed and replaced with 100 µl of fresh medium and 20 µl of Celltiter 96 Aqueous One Solution Cell Proliferation Assay solution. The cells were incubated at 37°C for 1 h before the absorbency was measured at 490 nm. In the assays using cell death inhibitors, 3-methyladenin, wortmannin, zVAD-fmk, and necrostatin 1 were diluted to proper concentrations in cell culture medium and added shortly before the cells were exposed to nanoparticles.The cytotoxic potential of the metal oxides was assessed using the MTS assay. A549, NCI-H1650 and CNE-2Z were seeded at a concentration of 1×105 cells/ml in 24-well plates and incubated for 12 h. Cells were then exposed to either 30 µg/ml CuO or 30 µg/ml SiO2 nanoparticles for 8 h. Samples were examined for apoptosis using the in situ cell death detection kit according to the manufacturer’s instruction manual.A549 cells were seeded at a concentration of 1×105 cells/ml in 24-well plates and incubated overnight, cells were treated with 30 µg/ml CuO or 30 µg/ml SiO2nanoparticles for 12 h. For autophagy flux analysis, A549 cells were seeded at a concentration of 5×105 cells/ml in 6-well plates and incubated overnight, cells were treated with 50 nM bafilomycin A1 for 1 hour before added 30 µg/ml CuO nanoparticles for 3 h. Cells were lysed with ice-cold lysis buffer , then the same volume of 2× Loading Buffer  were added, samples were splitted on ice for 15 min and then denatured at 95°C for 15 min. Samples were subjected to Western blot analysis, and band densities were analyzed using AlphaEaseFC or Quantity One software.For LC3 analysis, A549 cells were seeded at a concentration of 1×10A549 cells were treated with CuO (30 µg/ml), SiO2 NPs (30 µg/ml) or rapamycin (5 µM) for 12 h. Cells were then collected and fixed with 2.5% glutaraldehyde. Samples were then fixed in 1% OsO4 for 1 h, dehydrated by increasingconcentrations of acetone, and gradually infiltrated with epoxy resin. Ultra-thin sections were stained with uranyl acetate and lead citrate. The percentage of autophagy-positive cells was calculated. A cell showing two or more autophagosomes was defined to be an autophagy-positive cell.*P<0.05; **P<0.01.2 NPs (30 µg/ml) or rapamycin (5 µM) for 6 h at 37°C and 5% CO2. EGFP+ dots in the cell were counted via Olympus laser-scanning spectrum confocal system linked to a microscope. Images were captured under the 100× oil objective with the confocal acquisition software FV10-ASW 3.0. A cell containing three or more EGFP+ dots was defined as an LC3-positive cell.A549 cells were seeded on coverslips in 12-well plates, and 24 h later, transfected with LC3-EGFP plasmid. 24 h after transfection, cells were incubated with CuO (30 µg/ml), SiOA549 cells were seeded in a 24-well plate, and 24 h later, treated with control siRNA or ATG5-specific siRNA duplex (100 µg in 100 µl) in DEPC-treated saline. After 36 h, knockdown efficiency was determined by Western blotting with anti-Atg5 antibody. 36 h after transfection, cells were digested with trypsin and seeded on 96-well plates. 12 h later, CuO NPs (30 µg/ml) was added to the Atg5 siRNA and control siRNA group in the 96-well plate, and the MTS assay was conducted on the next day. In addition, siRNA-treated cells were also subject to Western blotting after CuO exposure for detection of LC3.p<0.001) (p<0.01), and all the other nanoparticles tested appeared to have no cytotoxic effect. Next, we evaluated the cytotoxicity of these nanoparticles in two other cell lines, NCI-H1650 and CNE-2Z, and found similar results -5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium), as an analog of MTT, can give a water-soluble formazan product in the presence of phenazinemethosulfate (PMS) and be used in the microculture screening assay for cell growth. The scale of Y-axis is the ratio of sample colored products absorbance at 490 nm vs. the control colored products absorbance at 490 nm. Therefore, the scale of Y-axis is proportional to the cell viability p<0.001)  and the p<0.001) . However results , Fig. 1D results . Even thCells can degrade proteins and organelles by engulfing them in double-membrane vacuoles known as autophagosomes, this process is called autophagy Although autophagy can cause cell death, it is also thought to be a survival mechanism for cells under certain conditions Copper oxide nanoparticles are widely used in many products, including antimicrobial agents, heat transfer fluids, semiconductors and healthcare products, such as intrauterine contraceptive devices CuO NPs appeared to have greater cytotoxicity compared to their bulk counterpart and to other metal oxide NPs Our study shows that copper oxide nanoparticles have a higher cytotoxic potential compared to the other metal oxide NPs used in this study, and that this cytotoxicity may involve the autophagic pathway. We also found in subsequent experiments that CuO NPs are also acutely toxic in mice, and cause significant pulmonary edema and death within 24 h. It has also been shown that copper NPs have acute toxicological effects in models such as the zebrafish and rat


Inflammation and hypoxia activate the fractalkine (CX3CL1) receptor (CX3CR1)-related signaling pathway. Tumor necrosis factor alpha (TNFα) induces CX3CL1, influencing a mechanism of CX3CL1 autoregulation by CX3CR1 expression. We compared spontaneous and lipopolysaccharide (LPS)-induced CX3CL1 and TNFα production by human placenta under normoxic vs. hypoxic conditions, with respect to CX3CR1 expression and its functional status.N = 24) were perfused extracorporeally. CX3CL1 and TNFα concentrations were measured in the perfusion fluid by ELISA. LPS, anti-CX3CR1 antibodies and pirfenidone were used in respective subgroups. After perfusion, CX3CR1 expression was estimated in placental tissue using quantitative immunohistochemistry, and the final results were adjusted for the mean microvascular density.Placental lobules of term placentae (p < 0.05). Unlike in normoxia, anti-CX3CR1 administration in hypoxia significantly reduced the LPS-evoked response. CX3CR1 expression was augmented by hypoxia and reached 260.9 ± 41 (% ±SEM) of the reference value in normoxia. Positive immunostaining for CX3CR1 corresponded to the vascular endothelium. Pirfenidone inhibited hypoxia + LPS-related increase in TNFα production and prevented the up-regulation of CX3CR1.The highest increase in CX3CL1 concentration in response to LPS was observed in hypoxia (The modulatory influence of TNFα on CX3CR1 expression in hypoxia and CX3CL1/CX3CR1 interaction may serve as a compensatory mechanism to preserve or augment the pro-inflammatory course of intercellular interactions in placental endothelium. Because placental vessels lack autonomic innervation, circulating and locally produced humoral factors must play a crucial role in communication between the compartments of the utero-placento-fetal unit . In addiData from studies on the role of CX3CL1 in reproduction are still being accumulated. It has been reported that, together with some other cytokines , CX3CL1 is involved in the processes of implantation, invasion of the trophoblast into the spiral uterine arteries, placental angiogenesis, responses to inflammatory and immunological factors in the utero-placental interface and the induction of labor –10.Interestingly, even during the course of normal pregnancy, the immunological status of the placental unit resembles, to some degree, a controlled inflammatory state , 12. ThuCX3CL1 in humans binds to a single Gαi protein-linked transmembrane receptor, CX3CR1 (previously known as V28), to express biological activity . CX3CR1 Changes in CX3CR1 expression may be important because autoregulatory interactions between CX3CL1 and CX3CR1 have been reported. It has been proposed that CX3CL1 induces its own expression via the PI3-kinase/PDK1/Akt/NIK/IKK/nuclear factor kappa beta (NF-κB) signaling pathway . Tumor nMany stimuli potentially able to disrupt cell homeostasis, including hypoxia, may induce CX3CL1 secretion , 18. ActEndothelial cells of the vascular system, vascular smooth muscle cells and amniotic epithelial cells are the main sources of CX3CL1 in the human placenta and membranes , 24. WhiThe aim of this study is to examine spontaneous and LPS-induced CX3CL1 and TNFα production by perfused human placental lobules in vitro with respect to CX3CR1 expression and functional status.N = 24; mean gestational age of 277 ± 6 days) were obtained from primigravidas after normal-course pregnancies. The placentae were delivered by elective cesarean sections and were dually perfused in vitro at 37 °C, using a modified Schneider’s method  delivered the currents to the two-channel biological oxygen monitor , connected to a computer-assisted data acquisition system.The single isolated placental lobules (two per placenta) were bilaterally perfused in vitro. A diagram of the two-sided closed perfusion system is shown in Fig. ® Human Fractalkine ELISA Kit  has a very high specificity that exceeds other available ELISA tests for the detection of CX3CL1 in placental perfusates, to the best of our knowledge. The samples were collected every 30 min from 30 to 150 min. The mean values for each group and at each time-point were calculated.During the 150 min of perfusion, including the initial 30-min adaptive phase, basal (immediately before administration of LPS) and LPS-evoked (10 ng/ml) CX3CL1 secretion into the fetal side placental circulation was examined quantitatively in perfusion fluid samples using ELISA. The RayBioThe dose of LPS used in this study was precisely titrated during the preliminary phase of the perfusion experiments. After a series of dose–response curve analyses we established 10 ng/ml as the mean dose of LPS eliciting 45–55 % maximal TNFα response in normoxic conditions (data not shown).Additionally, the TNFα levels in the perfusion fluid samples obtained at the same time-points were examined. Commercially available kits were used  following the manufacturer’s instructions. The minimum detectable dose of TNFα was <10 pg/ml.Experiments also tested the effect of CX3CR1 blockade on CX3CL1 secretion into the fetal side perfusion fluid under normoxic or hypoxic conditions with or without LPS administration. To block CX3CR1 on the fetal side of placental circulation, rabbit anti-human chemokine (C-X3C Motif) receptor 1 (CX3CR1) polyclonal “neutralizing” antibody  was administered via perfusion fluid  during the initial adaptive period of the respective perfusions. The final dilution of the antibody amounted to 1 mg/ml and was determined according to available datasheet protocols and experimental measurements. Dilution series were used followed by immunohistochemical staining of the perfused placental tissue specimens for CX3CR1 see “”, and coThe experimental setup for the perfusion procedures and the measurements at fixed time-points is presented in Fig. +) with parameters matching group II were perfused using the same procedure (hypoxic conditions), except that pirfenidone  was added into the perfusion fluid.Additionally, to investigate the influence of the anti-inflammatory compound pirfenidone, a non-selective inhibitor of TNFα production, another twelve isolated placental lobules  were prepared for each examined lobule. The methodology used in the applied in vitro experiment precluded obtaining the placental specimens during the phase of perfusion. Thus, we do not have collected data on CX3CR1 expressions at the same time-points as scheduled for CX3CL1 and TNFα. However, as a kind of control measurement, the tissue specimen of each perfused placental lobule was collected at the end of the adaptive phase for CX3CR1 immunostaining.To visualize CX3CR1, standard immunohistochemical procedures were used. Rabbit polyclonal antibody IgG to CX3CR1  was used as the primary antibody, and goat anti-rabbit IgG was used as the biotinylated secondary antibody . Visualization of the primary anti-receptor antibodies was performed using a StreptABComplex/HRP Duet  kit following the procedure recommended by the manufacturer with 3,3′-diaminobenzidine serving as the chromogen. The respective negative controls for immunostaining were prepared simultaneously by replacement of the polyclonal primary antibody with normal rabbit preimmune IgG diluted with PBS containing 3 % bovine serum albumin at the same protein concentration as that for the primary antibody.2, and the total number of preparations was 144 per group. Measurement of the total vascular area was performed. The total lumen area of all types of identified vessels was summed in both groups. To eliminate technical error caused by uniaxial sectioning of the vessel, the lowest value of Ferret’s diameter was accepted as the diameter for each lumen. Thus, V/EVTI represents the ratio which is most closely correlated with the intensity of vascularization.The vessels of the placental sections were identified with rabbit polyclonal antibody anti-CD31 . The tissue was incubated with the primary antibody for 30 min. A biotinylated goat anti-rabbit antibody (Abcam) was used as the secondary antibody. Next, morphometric analysis was performed using light microscopy with a Leica imaging workstation , and the vascular/extravascular tissular index (V/EVTI) was estimated in calibrated areas of the placental sections. Each preparation (paraffin section) underwent three area analyses repeated by two independent observers. The single area measured with the picture analysis method totaled 721,320 μm2 at 200× magnification. In each group, 144 visual fields were analyzed . To achieve maximum accuracy of the measurements, the following factors were controlled or monitored: average of image intake, hue, illumination, luminance, power supply, relation of illumination to quantification of the percentage area of positively stained structures, shading correction and warming up. A detailed description of these morphometric procedures has been provided previously  pg/ml, whereas in group II, the median was 81 (95 % CI 56–111) pg/ml (Table p < 0.05) were measured consistently throughout the 120-min perfusion time in hypoxic group IIA. With normoxia, the neutralizing anti-CX3CR1 antibody did not affect the production of CX3CL1. Thus, the differences in the mean CX3CL1 levels between subgroups IA and IB were not statistically significant. Unlike normoxic conditions, blockade of CX3CR1 with hypoxia produced evident disturbance of LPS-induced CX3CL1 release. Despite the increase from its initial values, the mean CX3CL1 concentrations at the respective time-points were lower in group IIB (p < 0.05) than in both group IIA and group I. As shown in Fig. The results pertaining to the CX3CL1 levels are shown in Fig. initTNFα) measured at the end of the adaptive phase  revealed that hypoxia increases the production of this cytokine in perfused placental lobules. The mean initTNFα in group I was 39.8 ± 16.8 pg/ml (±SEM), whereas for group II the average initTNFα was 72.4 ± 17.1 pg/ml. The medians, rounded to the nearest whole number, were 43 (95 % CI 34–117) pg/ml and 75 (95 % CI 69–160) pg/ml for group I and group II, respectively  may significantly increase the production of TNFα by endothelial cells, including the placental endothelium , 32, 35.According to our results, there is no reason to doubt that LPS and TNFα augment the production of chemokine CX3CL1. Similar results, including but not limited to LPS and TNFα production, were observed by other investigators in research concerning inflammatory cytokine signaling in endothelial cells , 22, 35.These contradictory results have created uncertainty regarding whether endothelium-derived CX3CL1 acts in an organ-, site- or state-dependent manner. Recent data indicate that chemokines have various effects on endothelial cells depending on their organ of origin , 39. MorThe mechanisms contributing to increased expression of CX3CR1 in hypoxic conditions may involve HIF-1-dependent and NF-κB-dependent signaling pathways, as shown in prostate cancer cells . FurtherCX3CL1 might play an important role in the pathophysiological process of inflammatory angiogenesis. The angiogenic activity of CX3CL1 required two signal pathways: RAF-1/MEK/ERK and PI3K/Akt/eNOS/NO. Interestingly, both pathways were mediated via CX3CR1 .+) and counteracted CX3CR1 overexpression.In endothelial cells and other cell types, TNFα acts through NF-κB to strongly induce both CX3CR1 expression and CX3CL1 production , 43. PirIn conclusion, these results indicate that, in hypoxia, interaction between the two important members of the placental cytokine network, TNFα and CX3CL1, may be influenced by increased TNFα levels. Up-regulation of CX3CR1 is needed for increased CX3CL1 production in hypoxia. TNFα-induced up-regulation of CX3CR1 expression is crucial and most likely overrides CX3CL1 autoregulation. Further studies on an in vivo placental perfusion model with blood-filled vessels is required for more definite conclusions, including potential treatment options in selected hypoxia- and hypoxemia-related inflammatory gestational pathologies.


Intensive insulin therapy (IIT) has been shown to reduce morbidity and mortality of critically ill patients ,2. A surThree ICUs developed and implemented an evidence-based guideline for IIT. For 1 year before and 1 year after implementation, all disposables and devices explicitly used for IIT were identified in each hospital. Local costs were calculated, based on costs for disposables and devices. Variable cost included costs associated with disposables. Fixed cost included costs associated with syringe pumps and point-of-care devices for blood glucose level (BGL) measurements.P < 0.001). The number of BGL measurements per patient per day doubled from 4 (3 to 7) to 9 (5 to 12) per day (P < 0.001). Yearly variable costs increased from €58.574 to €118.624 (P < 0.001), yearly fixed costs increased from €450 to €14.282 (P < 0.001). Importantly, costs differed substantially from one centre to another: variable costs per patient increased from €34 (€13 to 75) to €116 (€61 to 212) (P < 0.001), from €13 (€5 to 44) to €48 (€32 to 88) (P < 0.001) and from €15 (€7 to 34) to €31 (€15 to 70) (P < 0.001) for the three ICUs, respectively. Fixed costs per bed per year increased from €0 to €250 (P < 0.001), from €13 to €384 (P < 0.001) and from €25 to €544 (P < 0.001) for the three ICUs, respectively.A total of 2,490 patients were subjected to IIT. Patient demographics did not differ among the three ICUs and did not change over time. Median BGL declined from 119 (99 to 150) to 105 (85 to 130) mg/dl (Glucose control-associated costs rise with the implementation of IIT. Major differences in costs are noticed when comparing ICUs with similar patient cohorts and similar blood glucose control metrics after implementation of IIT.