Concentration-dependent Way Research Articles

Defensive Properties of Ginsenoside Re against UV-B-Induced Oxidative Stress through Up-Regulating Glutathione and Superoxide Dismutase in HaCaT Keratinocytes.

Ginseng is now used worldwide as a traditional Oriental medicine. Ginsenosides, also known as ginseng saponins, are responsible for most pharmacological efficacies of ginseng. This work aimed to assess the novel skin anti-photoaging potential of ginsenoside Re (Re), a protopanaxatriol-type ginsenoside, by analyzing reactive oxygen species (ROS), pro-matrix metalloproteinase-2 (proMMP-2) and -9 (proMMP-9), total glutathione (GSH), superoxide dismutase (SOD), and cellular viability in UV-B-irradiated HaCaT keratinocytes. When HaCaT cells were pretreated with Re prior to UV-B irradiation, Re significantly suppressed the UV-B-induced ROS elevation. It was also able to attenuate the UV-B-induced proMMP-2 and -9 elevations at both activity and protein levels. Re was capable of overcoming the UV-B-reduced total GSH content and SOD activity in concentration-dependent ways. Under the experimental conditions used, Re could interfere with cellular viabilities in neither non-irradiated nor UV-B-irradiated keratinocytes.

Soybean (Glycine max) prevents the progression of breast cancer cells by downregulating the level of histone demethylase JMJD5.

Breast cancer is the first noticeable disease in female patients. Long-term use of soybean (Glycine max) may prevent the progression of cancer. However, the molecular mechanism for the functions of soybean remains unclear. Histone demethylase JMJD5, an important epigenetic molecule, is overexpressed in the progression of breast cancer suggesting that soybean may ameliorate cancer by affecting the expression of JMJD5. To test the hypothesis, human breast cancer cell lines MCF-7 and MDA-MB-231 were treated with different concentrations of soybean and/or transfected with the plasmids pcDNA3.1-JMJD5 and pTZU6 + 1-shRNA-JMJD5. The growth rate was measured using xCELLigence real-time cell analysis. The level of JMJD5 was measured by using quantitative reverse transcription-polymerase chain reaction and Western blot. Soybean showed significant inhibitory effects on the growth rates ofMCF-7 and MDA-MB-231 cells in a concentration-dependent way (P < 0.05). Meanwhile, the levels of JMJD5 were reduced with the increase of soybean concentration (P < 0.05). JMJD5 transfection increased the growth rates of MCF-7 and MDA-MB-231 by 25% and 40%. In contrast, the growth rates of MCF-7 and MDA-MB-231 cells were decreased by 17% and 23% after being transfected with JMJD5 shRNA. Soybean inhibited the growth rate of MCF-7 and MDA-MB-231 cells when they were transfected by JMJD5 gene but no for the cells transfected with JMJD5 shRNA. The complicated compositions of soybean will be beneficial to the therapy of breast cancer since its causes may be involved in multiple aspects. Soybean represses breast cancer development by downregulating the level of JMJD5.

Exploring the Nanotoxicology of MoS2: A Study on the Interaction of MoS2 Nanoflakes and K+ Channels.

Molybdenum disulfide (MoS2) nanomaterial has recently found various applications in the biomedical field mainly due to its outstanding physicochemical properties. However, little is known about its interactions with biological systems at the atomic level, which intimately relates to the biocompatibility of the material. To provide insights into the effects of MoS2 in biological entities, we investigated the interactions of MoS2 with proteins from a functionally important membrane family, the ubiquitous potassium (K+) channels. Here, we study four representative K+ channels-KcsA, Kir3.2, the Kv1.2 paddle chimera, and K2P2-to investigate their interactions with a triangular MoS2 nanoflake using Molecular Dynamics (MD) simulations combined with electrophysiology experiments. These particular K+ channels were selected based on the diversity in their structure; that is, although these K+ channels display similar structural motifs, they also contain significant differences related to their particular function. Our results indicate that the MoS2 nanoflake is able to stably bind to three out of the four channels, albeit through distinct binding modes. The binding mode between each channel and MoS2 underlies the specific deleterious influence on the channel's basic physiological function: For KcsA, MoS2 binds on the extracellular loops, which indirectly destroys the delicate structure of the selectivity filter causing a strong leak of K+ ions. In the binding mode with Kir3.2, the MoS2 nanoflake completely covers the entrance to the channel pore affecting the normal ion conduction. For the Kv1.2 chimera, the MoS2 nanoflake prefers to bind into a crevice located at the extracellular side of the Voltage Sensor Domain (VSD). Interestingly, the crevice involves the N-terminal segment of S4, a crucial transmembrane helix which directly controls the gating process of the Kv1.2 chimera channel by electromechanical coupling the VSD to the transmembrane electric field. MoS2 in contact with S4 from the Kv1.2 chimera, potentially influences the channel's gating process from open to closed states. In all three systems, the van der Waals contribution to the total energy dominates the binding interactions; also, hydrophobic residues contribute the most contact points, which agrees with the strong hydrophobic character of the MoS2 nanomaterial. Electrophysiology recordings using two-electrode voltage-clamp show that currents of Kir3.2 and Kv1.2 are both blocked by the MoS2 nanoflakes in a concentration-dependent way. While the background K+ channel, K2P2 (TREK-1), identified as a negative control, is not blocked by the MoS2 nanoflakes. The large and rigid extracellular domain of K2P2 appears to protect the channel from disturbance by the nanoflakes. Intrinsic chemical properties of MoS2, together with the specific features of the channels, such as the electrostatic character and complex surface architecture, determine the critical details of the binding events. These findings might shed light on the potential nanotoxicology of MoS2 nanomaterials and help us to understand the underlying molecular mechanism.

Allelopathic effect of the rice straw aqueous extract on the growth of Microcystis aeruginosa

With the exploration and development of the practical technology for prevention and control of harmful algae blooms (HABs), cyanobacterium Microcystis aeruginosa (M. aeruginosa) blooms have been an urgent problem in the area of water resources protection. Allelopathic algicides are regarded as potential natural resources to constrain HABs for their exclusive and efficient characteristics. Previously, the allelopathic effect of rice has received great attention, but most of them are focused on herbicides to inhibit barnyard grass. In the present study, the effect of algal cells treatment with different concentrations of the rice straw aqueous extract were tested. A continuous culture system was established and the results suggested that the doses of rice straw aqueous extract in the range from 4.0 to 10.0gL−1 observably suppressed the growth of algae cells in a concentration-dependent way. After 9 days of treatment, the algae cells' metabolic activity decayed and the largest inhibition rate could be up to 98%, almost no algae growth was observed. In the case of eliminate the interference of nutrient elements, these results confirmed that rice straw aqueous extract may act as an algistatic agent.

ANTIOXIDANT AND HEPATOPROTECTIVE EFFECTS OF ASTRAGALUS ECHINOPS AND ASTRAGALUS LOGOPODIOIDES ETHANOLIC EXTRACTS ON PARACETAMOL-INDUCED LIVER INJURY IN RATS

Background: Paracetamol (PCM) has an adequate safety profile when taken in normal doses. However, it could&#x0D; produce oxidative stress with liver injury when taken in an overdose. Plants of Astragalus genus (F. Fabaceae) are of&#x0D; wide-spread applications. Astragalus echinops (A. echinops) and Astragalus logopodioides (A. logopodioides) were&#x0D; tested for their potential hepatoprotective activities against liver injury induced by PCM in rats.&#x0D; Material and Methods: Seven groups of rats were used for determination of hepatoprotective activities of the extracts.&#x0D; The normal and hepatotoxic control groups received the vehicle while other groups were treated with silymarin (100&#x0D; mg/ kg), A. echinops (250 and 500 mg/kg) and A. logopodioides (250 and 500 mg/ kg), respectively for seven days.&#x0D; Liver injury was induced on the 5th day by oral dosing of PCM (2g/kg) to all rats except those in normal control group.&#x0D; Moreover, the in vitro antioxidant activities of A. echinops and A. logopodioides extracts were tested using 2,2-&#x0D; diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging.&#x0D; Results: Hepatic enzyme markers as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and&#x0D; glutamyl transferase and level of total bilirubin were significantly elevated, while total protein and albumin were&#x0D; declined significantly in PCM-exposed animals. The liver antioxidant markers like the activities of superoxide&#x0D; dismutase, catalase and glutathione peroxidase and the levels of reduced glutathione were significantly declined, while&#x0D; hepatic malondialdehyde levels were significantly increased in PCM alone-treated rats. Administration of A. echinops&#x0D; (250 and 500 mg/kg) and A. logopodioides (500 mg/ kg) extracts prior to PCM, significantly protected against the&#x0D; elevation in the serum activities of hepatic enzymes and bilirubin and reduced oxidative stress. The hepatoprotective&#x0D; effect of both extracts was further confirmed by histological findings in the liver tissue. In addition, both extracts&#x0D; displayed in vitro antioxidant activities in a concentration-dependent way.&#x0D; Conclusion: Our results suggest that both extracts protect the liver against oxidative damage and they could be used as&#x0D; effective hepatoprotectives against PCM induced liver injury.

Kaempferol Alleviates the Interleukin-1β-Induced Inflammation in Rat Osteoarthritis Chondrocytes via Suppression of NF-κB.

BackgroundThis study was designed to examine the anti-inflammatory and anti-osteoarthritis (OA) effects of kaempferol in rat articular chondrocytes stimulated with interleukin-1β.Material/MethodsRat articular chondrocytes cultures were treated with interleukin-1β alone or with kaempferol (25, 50, 100, and 200 μM) and interleukin-1β. The effect of kaempferol on chondrocyte cells viability was measured by MTT assay. The effect on prostaglandin E2 (PGE2) and nitric oxide (NO) level were also assessed using the ELISA and Griess reagent, respectively, for kaempferol activity. Moreover, the expression of iNOS, Cox-2 and activation of NF-κB under influence of kaempferol was also assessed by Western blot.ResultsKaempferol treatment (up to 100 μM) in a concentration-dependent way caused reduction in the interleukin-1β-stimulated formations of PGE2 and NO. Kaempferol also upregulated the expression of iNOS and Cox-2 in interleukin-1β-stimulated rat OA chondrocytes. Additionally, kaempferol was found to inhibit the IκBα degradation and NF-κB activation in rat chondrocytes stimulated with interleukin-1β.ConclusionsKaempferol significantly caused reduction in interleukin-1β-stimulated pro-inflammatory mediators in rat OA chondrocytes by inhibiting the NF-κB pathway. These results suggest that kaempferol had significant anti-inflammatory and anti-arthritis effects. Thus, kaempferol, as a novel therapeutic active agent, may prevent, stop, or retard the progression of OA.

PDok2, caspase 3 dependent glioma cell growth arrest by nitidine chloride.

Nitidine chloride (NC) is known to exert anticancer and anti-metastatic effects on a variety of tumors. Recently, NC has also been shown to inhibit PIK3/AKT/mTOR axis in U87 human glioma cells. The study shows NC employing pDok2, caspase 3 dependent cell death in C6 rat glioma and U87 human malignant glioblastoma cells. The effect of NC on glioblastoma cell lines was accessed by MTT, clonogenic and wound healing assays. Cell cycle analysis was performed by FACS. Moreover, the effect of NC on downstream target proteins, such as caspase3, pDok2, PARP, and Gsk3 beta, were measured by western blotting. Overexpressed pDok2 protein has recently been reported as a prognostic marker with poor outcomes for human glioblastoma multiformae. We found that NC inhibits pDok2 in U87 cells in a concentration-dependent way. We further showed that cleaved PARP and cleaved caspase 3 protein expressions were increased in C6 cells treated with NC in a dose-dependent way. NC effectively attenuated C6 cells growth and colony formation at 8μM (micromoles) concentration. Cell cycle arrest in G2/M phase was further confirmed by flow cytometry. NC also exhibited its inhibitory effect on Gsk3 beta, which has been proven to be altered in glioma biology. Collectively, we predicted that NC could be employed as a potential anti-glioma mediator that needs attention to explore the mechanisms of its activity.

Cysteine as a potential anti-amyloidogenic agent with protective ability against amyloid induced cytotoxicity

Protein aggregation and misfolding have been allied with numerous human disorders and thus inhibition of such occurrence has been center for intense research efforts against these diseases. Here, we investigated anti-fibrillation activity of cysteine and its effect on kinetics of stem bromelain amyloid fibril formation. We established the anti-fibrillation and anti aggregation activities of cysteine by using multiple approaches like turbidity measurements, dye binding assays (ThT and ANS) and structural changes were monitored by circular dichroism (CD) followed by electron microscopy. Our experimental study inferred that cysteine inhibits temperature induced fibrillation of protein in a concentration dependent way. In addition, MDA-MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as compared to the fibrils alone. Furthermore, dynamic light scattering studies of native, aggregated as well as incubated (amyloids in presence of cysteine) samples indicates that cysteine restores native like structures of stem bromelain. Isothermal titration calorimetric results revealed that hydrogen bonding between cysteine and stem bromelain plays a significant role during inhibition of stem bromelain aggregation. However, thiophilic interaction between thiol group of cysteine and aromatic amino acid residue of stem bromelain may also have noteworthy role in inhibition of amyloid formation.

The vitamin B6 paradox: Supplementation with high concentrations of pyridoxine leads to decreased vitamin B6 function

Vitamin B6 is a water-soluble vitamin that functions as a coenzyme in many reactions involved in amino acid, carbohydrates and lipid metabolism. Since 2014, >50 cases of sensory neuronal pain due to vitamin B6 supplementation were reported. Up to now, the mechanism of this toxicity is enigmatic and the contribution of the various B6 vitamers to this toxicity is largely unknown.In the present study, the neurotoxicity of the different forms of vitamin B6 is tested on SHSY5Y and CaCo-2 cells. Cells were exposed to pyridoxine, pyridoxamine, pyridoxal, pyridoxal-5-phosphate or pyridoxamine-5-phosphate for 24h, after which cell viability was measured using the MTT assay. The expression of Bax and caspase-8 was tested after the 24h exposure. The effect of the vitamers on two pyridoxal-5-phosphate dependent enzymes was also tested.Pyridoxine induced cell death in a concentration-dependent way in SHSY5Y cells. The other vitamers did not affect cell viability. Pyridoxine significantly increased the expression of Bax and caspase-8. Moreover, both pyridoxal-5-phosphate dependent enzymes were inhibited by pyridoxine.In conclusion, the present study indicates that the neuropathy observed after taking a relatively high dose of vitamin B6 supplements is due to pyridoxine. The inactive form pyridoxine competitively inhibits the active pyridoxal-5′-phosphate. Consequently, symptoms of vitamin B6 supplementation are similar to those of vitamin B6 deficiency.

Interactions of a prenylated flavonoid from Dalea elegans with fluconazole against azole- resistant Candida albicans

BackgroundThe prenylated flavonoid 2′, 4′-dihydroxy-5′-(1′″, 1′″-dimethylallyl)-8-prenylpinocembrin (8PP, formerly 6PP) shows antifungal activity, inhibits rhodamine 6G efflux and reverses fluconazole (FCZ) resistance in azole-resistant Candida albicans overexpressing cdr1, cdr2 and mdr1 transporters. Purpose and designIn this paper, we tried to characterize 8PP in vitro interactions on the cell growth and lethality of C. albicans. We also initiated preliminary in vivo toxicological studies on mice. MethodsThe effects of 8PP and FCZ on cell growth and viability of C. albicans were evaluated by CLSI guidelines. The checkerboard assay was used to search for interactions on cell growth. The time-kill assay was used to study fungicidal effects. Acute toxicity was evaluated at a single dose schedules. ResultsFrom the checkerboard design, and using a starting inoculum of 103CFU/ml, the fractional inhibitory concentration (FIC) of FCZ and 8PP could be determined as 0.11 and 0.50, respectively, with a FIC index value (FICI) of 0.61. This FICI and the isobologram showing a concave shape suggests an additive interaction between them. At a higher starting inoculum (105CFU/ml), C. albicans growth and viability were decreased by FCZ, 8PP and their combination in a concentration-dependent way. For FCZ, minimum fungicidal concentration (MFC) and FC50 (the concentration that kills 50% of the fungal cells) were 4-fold reduced (280–70µM) in combination with 125µM 8PP. A decrease of 3 log units in viable counts with respect to control was reached (3.65 ± 1.05 ‰, p< 0.0001). Thus, both fungistatic compounds when combined achieved an almost complete fungicidal effect at lower concentrations respecting of each of them alone. In preliminary toxicological assessment, lethal dose 50% (LD50) for 8PP by the i.p. route was 357 and 245mg/kg, for female and male adult albino mice, respectively. FCZ LD50 was 785 and 650mg/kg for female and male animals, respectively ConclusionsIn vitro results suggest additive interactions between 8PP and FCZ with respect to C. albicans cell growth. Besides killing per se, 8PP helps FCZ to achieve an almost complete fungicidal effect, which would be crucial to eradicate fungal infections.

Crystal structures, electrochemical properties, antioxidant screening and in vitro cytotoxic studies on four novel Cu(II) complexes of bidentate Schiff base ligands derived from 2-methoxyethylamine

Four novel Schiff base ligands and their copper(II) complexes, [Cu(L1)2] (1), [Cu(L2)2] (2), [Cu(L3)2] (3), and [Cu(L4)2] (4), were synthesized and characterized by elemental analyses, FT-IR, and UV–Vis spectroscopy. The ligands were synthesized from the condensation of 2-methoxyethylamine with various salicylaldehyde derivatives (x-salicylaldehyde for HLn, x = H (n = 1), 5-Br (n = 2), 3-OMe (n = 3), and 4-OMe (n = 4)). The molecular structures of 1, 2, and 3 were determined by the single crystal X-ray diffraction technique. The redox behavior studies of the complexes in acetonitrile display the electronic effects of the groups on the redox potential. The antioxidant activity of the Schiff base ligands and their Cu(II) complexes was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method and FRAP assay. Furthermore, the in vitro anticancer activity of compounds was screened, including MTT and migration assays against gastric cancer cell line (MKN-45). The results show that all ligands and complexes have antioxidant and anticancer activity in a concentration-dependent way.

Evaluating the wound healing ability of Melicope pteleifolia (Champ. Ex Benth.) T.g. Hartley

Melicope pteleifolia (Champ. ex benth.) T.g. Hartley, a folk medicinal plant, is used by ethnic minorities in Bidoup–Nui Ba National Park, Lam Dong Province, Vietnam to treat effectively wound, inflammation and skin ulcer. To scientifically prove the claimed utilization and understand the mechanism of action of the plant, the in vitro and in vivo healing properties of the extract and fractions of the plant were investigated. The ethanol 70 % extract (50 – 400 mg/mL), aqueous (200 mg/mL), ethyl acetate (100 mg/mL) and petroleum ether (50 mg/mL) fractions were used to evaluate the antibacterial activities by using agar diffusion method. The healing properties were in vitro investigated through fibroblasts and keratinocytes proliferation and migration (7.8 g/mL to 250 g/mL in accordance with each extract and fraction). Besides, the macrophage-induced inhibition of the nitric oxide (NO) production was examined (15.6 – 62.5 g/mL). In addition, the excision wound model was used to test the wound healing activity on mice model. We found that the ethanol extract and the ethyl acetate fraction showed potent activity against Staphylococcus aureus, Enterococcus feacalis and Pseudomonas aeruginosa. The extract and fractions stimulated fibroblasts and keratinocytes proliferation in a concentration-dependent way. They also inhibited macrophage produce NO. In addition, mice treated by the extract formed scabs on wound excision of mice model faster than the control group. The wound healing efficiency seems to involve antibacterial, stimulating fibroblasts and keratinocytes proliferation, inhibition of macrophages produce NO.

Hexachlorobenzene alters cell cycle by regulating p27-cyclin E-CDK2 and c-Src-p27 protein complexes

Hexachlorobenzene (HCB) is an organochlorine pollutant widely distributed in the environment around the entire world. Previous reports from our group and others have demonstrated that this compound is as an endocrine disruptor. We have also reported that HCB presents a co-carcinogenic effect in N-Nitroso-N-methyl-urea–induced mammary tumours in rats. In this work, we studied the effects of HCB on cell cycle progression and cell cycle regulating protein expression in the estrogen-sensitive breast cancer cell line, MCF-7. Here, we show that HCB alters cell cycle in a concentration-dependent way. The lowest assessed concentration (0.005μM) promotes the cell cycle progression, enhances cyclin D1 expression, and reduces the nuclear localization of p27 accompanied by an increased interaction between p27 and c-Src kinase. On the other hand, 5μM HCB delays the cell cycle progression and promotes the formation of the cyclin E-CDK2-p27 protein complex. Our results show that HCB stimulates cell proliferation through cell cycle modulation and c-Src involvement in MCF-7 cells. Here, we report for the first time that differential mechanisms of action of HCB on mammary cell cycle progression are triggered at different concentrations of this pollutant.

Intracellular trafficking and cellular uptake mechanism of PHBV nanoparticles for targeted delivery in epithelial cell lines

BackgroundNanotechnology is a science that involves imaging, measurement, modeling and a manipulation of matter at the nanometric scale. One application of this technology is drug delivery systems based on nanoparticles obtained from natural or synthetic sources. An example of these systems is synthetized from poly(3-hydroxybutyrate-co-3-hydroxyvalerate), which is a biodegradable, biocompatible and a low production cost polymer. The aim of this work was to investigate the uptake mechanism of PHBV nanoparticles in two different epithelial cell lines (HeLa and SKOV-3).ResultsAs a first step, we characterized size, shape and surface charge of nanoparticles using dynamic light scattering and transmission electron microscopy. Intracellular incorporation was evaluated through flow cytometry and fluorescence microscopy using intracellular markers. We concluded that cellular uptake mechanism is carried out in a time, concentration and energy dependent way. Our results showed that nanoparticle uptake displays a cell-specific pattern, since we have observed different colocalization in two different cell lines. In HeLa (Cervical cancer cells) this process may occur via classical endocytosis pathway and some internalization via caveolin-dependent was also observed, whereas in SKOV-3 (Ovarian cancer cells) these patterns were not observed. Rearrangement of actin filaments showed differential nanoparticle internalization patterns for HeLa and SKOV-3. Additionally, final fate of nanoparticles was also determined, showing that in both cell lines, nanoparticles ended up in lysosomes but at different times, where they are finally degraded, thereby releasing their contents.ConclusionsOur results, provide novel insight about PHBV nanoparticles internalization suggesting that for develop a proper drug delivery system is critical understand the uptake mechanism.

Development of the Electrochemical Detection System Using the Combination of Aptamer and Enzyme

Introduction Aptamers are DNA or RNA molecules that bind to targets (proteins, small compounds, cells etc.) with high affinity and specificity. In particular, DNA aptamers have attractive properties for construction of biosensors; they are chemically-synthesized and easy to be modified by functional group with low production cost. Aptamer-introduced materials have been utilized to develop numerous biosensors, and indeed, proteomic microarray-based platform using modified aptamers is commercially available. Due to its several useful properties, we have employed DNA aptamers for development of electrochemical biosensors. We focused on glucose dehydrogenases (GDHs), enzymes have been already adopted by industries and utilized in commercially available glucose monitoring systems, as signal transducers for electrochemical detection of targets by aptamers. In addition, it is well-known that thrombin cleaves substrate peptide conjugated electroactive compound, and aptameric enzyme subunit (AES), a homogeneous detection method based on enzymatic activity, was developed utilizing thrombin in our laboratory. Thus, we are developing electrochemical measurement platform for AES based on thrombin activity. In this presentation, we describe electrochemical target detection systems based on GDH-labeled DNA aptamers and AES utilizing thrombin. Methods 1. Electrochemical detection of targeted molecules using GDH-labeled aptamers We developed GDH-employed electrochemical aptasensors with three method of GDH-labeling to DNA aptamer; (i) avidin-biotin interaction1,2, (ii) GDH-binding aptamers3,4 and (iii) GDH-fused zinc finger protein (unpublished result, but the enzyme-labeling method using enzyme-fused zinc finger protein has been already reported5.) DNA aptamers were labeled with recombinant PQQGDH from Acinetobacter calcoaceticus or FADGDH from Aspergillus flavus. GDH-fused zinc finger protein was expressed in Escherichia coli BL21(DE3) and purified by affinity chromatography. Thiolated aptamers or biotinylated antibody were immobilized on gold electrode. After incubation with target molecules, the gold electrode was immersed into GDH-labeled aptamer solution. We measured the response current to addition of glucose and m-PMS. 2. Electrochemical measurement of thrombin activity via interdigitated array electrodes We aim at measurement of target molecules by using interdigitated array (IDA) electrodes. IDA electrodes are suitable for redox cycling, which lead to achieve highly sensitive detection with a combination of a redox agent. Therefore, we measured thrombin activity using substrate peptide conjugated p-aminophenol. p-aminophenol is characterized as a redox agent. First, thrombin activity without inhibition for p-aminophenol-conjugating peptide was measured by IDA electrodes and the increased sensitivity via redox cycling was confirmed. Next, the activity with inhibition was investigated. Results and discussion 1. We found significant decreasing enzymatic activity of PQQGDH conjugated with streptavidin, but measurement of thrombin was attained and the detection threshold was indicated as 40 nM due to high catalytic efficiency of original PQQGDH. GDH fusing zinc finger protein showed 50% of enzymatic activity compared to the wild type GDH. DNA aptamer binds to GDHs did not affect the enzymatic activity. Sandwich-type sensing platform was utilized for protein detection, and target measurement based on the structural change was utilized for detection of adenosine. We successfully detected 500 pM-15 nM of VEGF and 0.2-5 mM of adenosine, respectively. 2. We observed changes in the electric current on addition of the electroactive substrate in the thrombin concentration dependent way. Under the redox cycling condition, level of the current was twice as high as the usual, non-redox cycling condition. Inhibition of thrombin was also found by addition of DNA aptamers for thrombin. These results suggested that a combination of electrochemical measurement of thrombin activity with IDA electrodes and thrombin inhibition system utilizing AES has the potential to achieve simplified diagnostic method in the future. Acknowledgement A part of this work was supported by AMED-SENTAN program.

Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells.

Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200μM DMF, respectively. After 24h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-L-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P < 0.05). The apoptotic HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P < 0.05). Meanwhile, loss of △Ψm, increase in reactive oxygen species and O2·-, and the decrease in catalase activity and glutathione (GSH) level were found after DMF treatment (P < 0.05). All these changes were significantly attenuated and even completely disappeared by adding NAC (P < 0.05). In conclusion, the cytotoxicity of DMF on cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.

Effect of flavopiridol on the proliferation, invasiveness and apoptosis of human prostatic cancer cell line LNCaP

Objective To investigate the effect of flavopiridol on the proliferation, invasiveness and apoptosis of human prostate cancer cell line LNCaP, and to explore the possibility of its application in clinical treatment. Methods MTT assay was used to detect cell proliferation, cell invasion in vitro was detected by Transwell assay, and flow cytometer was used to observe apoptosis. Results Flavopiridol inhibited the growth of LNCaP cells in a concentration-dependent and time-dependent way (P < 0.05), and reduced the ability of invasion capacity. After treated by 10 nmol/L flavopiridol for 24 h, the apoptosis rate was increased significantly to (7.5±0.9) % compared with the control group [(5.3±0.5) %] (P < 0.05). Conclusion Flavopiridol can inhibit proliferation of LNCaP cells and induce apoptosis, which may be applicable for the treatment of prostate cancer. Key words: Prostatic neoplasms; Flavopiridol; Proliferation; Apoptosis; Invasiveness

Mechanistic study of the inhibitory activity of Geum urbanum extract against α-Synuclein fibrillation

The presence of Lewy bodies and Lewy neurites is a major pathological hallmark of Parkinson's disease and is hypothesized to be linked to disease development, although this is not yet conclusive. Lewy bodies and Lewy neurites primarily consist of fibrillated α-Synuclein; yet, there is no treatment available targeting stabilization of α-Synuclein in its native state. The aim of the present study was to investigate the inhibitory activity of an ethanolic extract of Geum urbanum against α-Synuclein fibrillation and examine the structural changes of α-Synuclein in the presence of the extract. The anti-fibrillation and anti-aggregation activities of the plant extract were monitored by thioflavin T fibrillation assays and size exclusion chromatography, while structural changes were followed by circular dichroism, Fourier transform infrared spectroscopy, intrinsic fluorescence, small angle X-ray scattering and electron microscopy. Since the extract is a complex mixture, structure-function relationships could not be determined. Under the experimental conditions investigated, Geum urbanum was found to inhibit α-Synuclein fibrillation in a concentration dependent way, and to partly disintegrate preformed α-Synuclein fibrils. Based on the structural changes of α-Synuclein in the presence of extract, we propose that Geum urbanum delays α-Synuclein fibrillation either by reducing the fibrillation ability of one or more of the aggregation prone intermediates or by directing α-Synuclein aggregation towards a non-fibrillar state. However, whether these alterations of the fibrillation pathway lead to less pathogenic species is yet to be determined.

Effects of allocryptopine on outward potassium current and slow delayed rectifier potassium current in rabbit myocardium.

ObjectiveAllocryptopine (ALL) is an effective alkaloid of Corydalis decumbens (Thunb.) Pers. Papaveraceae and has proved to be anti-arrhythmic. The purpose of our study is to investigate the effects of ALL on transmural repolarizing ionic ingredients of outward potassium current (Ito) and slow delayed rectifier potassium current (IKs).MethodsThe monophasic action potential (MAP) technique was used to record the MAP duration of the epicardium (Epi), myocardium (M) and endocardium (Endo) of the rabbit heart and the whole cell patch clamp was used to record Ito and IKs in cardiomyocytes of Epi, M and Endo layers that were isolated from rabbit ventricles.ResultsThe effects of ALL on MAP of Epi, M and Endo layers were disequilibrium. ALL could effectively reduce the transmural dispersion of repolarization (TDR) in rabbit transmural ventricular wall. ALL decreased the current densities of Ito and IKs in a voltage and concentration dependent way and narrowed the repolarizing differences among three layers. The analysis of gating kinetics showed ALL accelerated the channel activation of Ito in M layers and partly inhibit the channel openings of Ito in Epi, M and Endo cells. On the other hand, ALL mainly slowed channel deactivation of IKs channel in Epi and Endo layers without affecting its activation.ConclusionsOur study gives partially explanation about the mechanisms of transmural inhibition of Ito and IKs channels by ALL in rabbit myocardium. These findings provide novel perspective regarding the anti-arrhythmogenesis application of ALL in clinical settings.

Oligoesculin fraction induces anti-tumor effects and promotes immune responses on B16-F10 mice melanoma.

Laccase was used to enzymatically polymerize esculin. Oligoesculin fraction was obtained after ultrafiltration through a 5-kDa membrane. Several studies have been carried out to prove the effectiveness of natural substances such as immunomodulators to promote the anti-cancer activity in situ. The purpose of our report was to explore whether the anti-tumor potential of the oligoesculin fraction in vitro and in vivo is linked to its immunological mechanisms in melanoma-bearing mice. We revealed that oligoesculin fraction reduced B16-F10 proliferation and migration in vitro in a dose-related manner. Moreover, melanin synthesis and tyrosinase activity were inhibited in these melanoma cells in a concentration-dependent way. The anti-tumor potential of oligoesculin fraction was also assessed in vivo. Our results showed that intraperitoneal administration of oligoesculin fraction, at 50mg/kg body weight (b.w.) for 21days, reduced tumor size and weight with percentages of inhibition of 94 and 87%, respectively. Oligoesculin fraction was effective in promoting lysosomal activity and nitric oxide (NO) production by peritoneal macrophages in tumor-implanted mice. In addition, the activities of natural killer (NK), cytotoxic T lymphocytes, and macrophages were significantly enhanced by oligoesculin fraction. These findings suggested that this polymer with its anti-tumor and immunomodulatory properties could be used for the treatment of melanoma.