Ecstasy metabolites and monoamine neurotransmitters upshift the Na+/K+ ATPase activity in mouse brain synaptosomes.

3,4-Methylenedioximethamphetamine (MDMA; "ecstasy") is a psychotropic drug with well-known neurotoxic effects mediated by hitherto not fully understood mechanisms. The Na+- and K+-activated adenosine 5'-triphosphatase (Na+/K+ ATPase), by maintaining the ion gradient across the cell membrane, regulates neuronal excitability. Thus, a perturbation of its function strongly impacts cell homeostasis, ultimately leading to neuronal dysfunction and death. Nevertheless, whether MDMA affects the Na+/K+ ATPase remains unknown. In this study, we used synaptosomes obtained from whole mouse brain to test the effects of MDMA, three of its major metabolites [α-methyldopamine, N-methyl-α-methyldopamine and 5-(glutathion-S-yl)-α-methyldopamine], serotonin (5-HT), dopamine, 3,4-dihydroxy-L-phenylalanine (L-Dopa) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the Na+/K+ ATPase function. A concentration-dependent increase of Na+/K+ ATPase activity was observed in synaptosomes exposed to the tested compounds (concentrations ranging from 0.0625 to 200 µM). These effects were independent of protein kinases A and C activities. Nevertheless, a rescue of the compounds' effects was observed in synaptosomes pre-incubated with the antioxidant N-acetylcysteine (1 mM), suggesting a role for reactive species-regulated pathways on the Na+/K+ ATPase effects. In agreement with this hypothesis, a similar increase in the pump activity was found in synaptosomes exposed to the chemical generator of superoxide radicals, phenazine methosulfate (1-250 µM). This study demonstrates the ability of MDMA metabolites, monoamine neurotransmitters, L-Dopa and DOPAC to alter the Na+/K+ ATPase function. This could represent a yet unknown mechanism of action of MDMA and its metabolites in the brain.

Protective effects of small heat shock proteins in Daphnia magna against heavy metal exposure.

Daphnia magna is one of the most commonly used model organisms to assess toxicity of heavy metal and other xenobiotics. However, the lack of knowledge about important stress-resistant molecules limits our understanding of the alteration of phenotypic and physiological traits of D. magna upon stress exposures. In this study, we focused on a chaperone family of small heat shock protein (sHSP) that has been found in archaea, bacteria and eukaryotes and plays an important role in stress tolerance. A total of eleven sHSP genes (termed DmsHSP1 - DmsHSP11) were identified from the D. magna genome, whose expression profiles during exposure to heavy metal (Cd2+, Cu2+ and Zn2+) and a few other potential pollutants were evaluated via qRT-PCR and RNA-Seq analysis. The results highlighted the predominant role of DmsHSP1 with the highest basal expression level in adults and robust upregulation upon exposure to heavy metals (Cu2+ > Cd2+ > Zn2+). In vivo, recombinant protein rDmsHSP1-21 and rDmsHSP11-12.8 could not only prevent model substrates agglutination induced by heavy metals or reducer dithiotreitol (DTT), but also protect tissue proteins and enzymes from denaturation and inactivation caused by heavy metals or high temperature. Ectopically expression of DmsHSP1-21 or DmsHSP11-12.8 in E. coli conferred host enhanced resistance against various abiotic stresses including Cd2+, Cu2+ and phenazine methosulfate (PMS). Knockdown of DmsHSP1-21 by RNAi, but not for DmsHSP11-12.8, significantly increased the vulnerability of D. magna to heavy metal exposure. Our work provides systematic information on the evolution and function of sHSPs in D. magna and leads to important insights into the mechanisms by which D. magna survive in adverse environments.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Determination of L-glutathione by spot test and spectrophotometric methods based on its interaction with phenazine.

This research paper presents simple and quick eco-friendly spot test and spectrophotometric methods for the determination of L-glutathione. The spot test assay is based on the formation of a color complex with phenazine methosulphate and L-glutathione on a thin-layer chromatography plate followed by image analysis using a scanner as a detector. For analysis, the image was converted into red, green, and blue (RGB) histograms. A series of parameters that influenced the color formation were investigated, and under the optimal conditions, a good linearity was observed in the range of 200-1000 µg mL-1 and 249-1000 µg mL-1 of L-glutathione with correlation coefficients of 0.9907 for B and 0.9903 for G channels. For the spectrophotometric method, a good linearity was obtained in the range of 2.1-60 µg mL-1 of L-glutathione concentration with a correlation coefficient of 0.9961. A mechanism of the reaction of L-glutathione with phenazine was proposed and confirmed by Fourier transform infrared and mass spectroscopy.

Optimization of a Colorimetric Assay to Determine Lactate Dehydrogenase B Activity Using Design of Experiments.

Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been considered as a potential target to treat these types of cancer. Herein, we propose a straightforward incomplete factorial (IF) design composed of 12 combinations of two reaction buffers, three pH values, three salt (NaCl) concentrations, and three incubation times, which we called IF-BPST (Buffer/pH/Salt/Time), for the optimization of a colorimetric LDH-B assay in a final volume of 100 µL using 96-well plates. The assay is based on the absorbance change at ~570 nm and the color change of the reaction mixture due to the release of NADH that reacts with nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), resulting in the formation of a blue-purple formazan. The results obtained using the IF-BPST were comparable with those obtained by response surface methodology. Our work revealed that the NBT/PMS assay with some modifications can be used to measure the activity of LDH-B and other dehydrogenases in a high-throughput screening format at the early stages of drug discovery. LDH-B containing lysates cannot be assayed directly, however, due to the sensitivity of the method toward detergents. Thus, we suggest precipitating the proteins in the lysates to remove the interfering detergents, and then to dissolve the protein pellet in a suitable buffer and carry out the assay.

Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis.

The level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli.

High-pressure tuning of primary photochemistry in bacterial photosynthesis: membrane-bound versus detergent-isolated reaction centers.

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multi-exponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states. Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster's Blue in methanol dehydrogenase assays.

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

Phenazine methosulphate-treated red blood cells activate NF-κB and upregulate endothelial ICAM-1 expression.

Although enhanced Red Blood Cell (RBC) - Endothelial Cell (EC) interaction, as well as RBC induced EC activation, have been extensively studied in several RBC-linked pathologies, the specific individual effects of oxidatively modified RBC on EC activation has not yet been documented. However, increasing evidence in both experimental and clinical studies suggests that oxidatively modified RBC could be considered potential pathogenic determinants in several acute and chronic diseases displaying systemic oxidative stress. Therefore, the present study aimed to explore the specific effects of oxidized RBC interaction with endothelial cells on intracellular signaling pathways that promote EC activation. RBC were exposed to oxidative stress induced by phenazine methosulphate (PMS). It is shown that the interaction of oxidatively modified RBC with cultured human umbilical vein endothelial cells (HUVEC) results in: a) EC activation as indicated by the increased surface expression of intercellular adhesion molecule -1 (ICAM-1); b) the activation of transcription factor NF-κB, an indicator of cellular oxidant stress. These results emphasize the specific contribution of oxidatively modified RBC interaction to EC activation and their possible pathological role in vascular diseases and oxidative stress.

A fast, sensitive, single-step colorimetric dipstick assay for quantifying ascorbic acid in urine.

The presence of ascorbate in human urine has been shown to be a useful dietary, fruit or vitamin C intake biomarker. More recently it has been discovered that ascorbate levels in urine can be used to facilitate the detection of precancerous colorectal polyps. While there are a number elaborate HPLC, MS or multi-step enzymatic "kit" methods to detect and quantify urinary ascorbate, these are time consuming and expensive. There are also a number of low-cost paper-based ascorbate detection dipsticks. However, the limits of detection and quantification accuracy for these dipsticks are not adequate for applications with human urine. To address these limitations, we have developed a fast, sensitive, single-step colorimetric assay that can be used to quantify ascorbate in urine and other biological fluids. The assay uses the tetrazolium salt, methylthiazolyldiphenyl-tetrazolium bromide (MTT), with the electron carrier phenazine methosulfate (PMS), in a chelated acidic phosphate-buffer to produce a vivid purple color in the presence of ascorbate. Confirmation of the performance of the assay and of its standard curve in human urine was also done using independent LC-MS/MS and NMR analyses. The lower limit of detection of the ascorbate dipstick assay described here was found to be 3.2 µM. The paper dipsticks are stable over a wide range of temperatures and can be stored for up to 150-days.

A salting-out assisted liquid-liquid microextraction procedure for determination of cysteine followed by spectrophotometric detection.

A new analytical method for sensitive determination of cysteine based on its interaction with phenazine methosulfate was developed using salting-out liquid-liquid microextraction followed by spectrophotometric detection. The mechanism of the reaction was studied and confirmed by Fourier transform infrared and mass spectroscopy. Experimental parameters affecting the extraction efficiency were investigated and under the optimal conditions, good linearity was observed in the range 0.2 - 6.0 µg mL-1 with a correlation coefficient of 0.9972. The limit of detection and limit of quantification were found to be 0.07 and 0.21 µg mL -1, respectively. The enrichment factor was 25. The developed methodology was applied for analysis of cysteine in food supplements. The obtained data were in good agreement with LC-MS/MS analysis.

Redox Modulating Factors Affect Longevity Regulation in Rotifers.

Rotifers are microinvertebrate models to study the phylogenetically based mechanisms of aging. Our study aimed to develop a physiological system with electron deprivation via a chemical electron carrier/acceptor pair together with extreme caloric restriction (ECR). Middle-aged Philodina acuticornis rotifers were treated with combinations of phenazine methosulfate (PMS, electron carrier) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT, electron acceptor) for a period of 72 hours under total food deprivation (preselection). The ability of XTT to be reduced was confirmed both in vitro (with NADH) and in vivo (with live rotifers). Subsequently, the respective electron acceptor alone at a lower dose was administered in combination with ECR for several months on preselected survivors. We found that the longevity of rotifers markedly increased (4×) after PMS/XTT/total food deprivation preselection followed by XTT/ECR treatment. Ascorbic acid in equivalent concentrations caused similar but less pronounced tendencies. The synergistic effect of chemical electron deprivation and ECR caused delayed aging and the development of an outstanding phenotype that we refer to as "super rotifers," characterized by increased longevity and retained reproductive ability compared with normal middle-aged individuals. The presented model provides new insights into the connection between redox modulation and age-related features in vivo.

A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency.

Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age-related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh-frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase-deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels - this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX-deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell-by-cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.

Addressed immobilization of biofunctionalized diatoms on electrodes by gold electrodeposition.

Diatoms are single cell microalgae with a silica shell (frustule), which possess a micro/nanoporous pattern of unparalleled diversity far beyond the possibilities of current micro- and nanofabrication techniques. To explore diatoms as natural three-dimensional nanostructured supports in sensing and biosensing devices, a simple, rapid and stable method to immobilize diatoms via gold electrodeposition is described. In this process, gold microstructures are formed, immobilizing diatoms by entrapment or crossing their nanopores. Varying the applied potential, time and HAuCl4 concentration, gold deposits of different morphologies and roughness are obtained, thereby determining the diatom immobilization process. Optical and scanning electron microscopy have been used to characterize diatom immobilization yields, the morphology of the gold microstructures, and the morphological integrity of diatoms. Cyclic voltammetry has been performed to characterize the gold deposits and to demonstrate the enhanced electrocatalytic activity of the gold-diatom electrodes. Electro-addressed immobilization of different diatoms on specific bands of interdigitated electrode arrays has been achieved, highlighting the potential application of diatoms for site-specific immobilization on microarrays. The feasibility to combine tailored immobilization with diatom biofunctionalization has also been demonstrated. Antibody-functionalized diatoms were immobilized on electrodes retaining their ability to detect its cognate antigen. The reported method exploits the natural three-dimensional nanostructures of diatoms together with their easy modification with biomolecules and the simplicity of gold electrodeposition to produce micro/nanostructured and highly electrocatalytic electrodes, providing low-cost and eco-friendly platforms and arrays with potential application in biosensing devices.

Serine racemase inhibition induces nitric oxide-mediated neurovascular protection during cerebral ischemia.

There are no effective neuroprotectant drugs for acute cerebral ischemia. Serine racemase (SR) synthesizes d-serine, which is involved in N-methyl-d-aspartate (NMDA) receptor-induced neurotoxicity. Recently, SR deletion was reported to protect against focal cerebral ischemia. However, regulatory mechanisms controlling SR-activity in the neurovascular unit (NVU) during cerebral ischemia remain to be clarified. We investigated the effects of SR inhibition on neurovascular protection after ischemia. The SR inhibitor phenazine methosulfate (PMS) alleviated neuronal damage in an ex vivo ischemic model (oxygen glucose deprivation [OGD]) using primary neuronal cultures, and in an in vivo mouse model of ischemia (middle cerebral artery occlusion [MCAO]). Ischemic preconditioning (IP) and PMS-treatment inhibited SR phosphorylation after ischemia ex vivo. In addition, SR phosphorylation after MCAO was also decreased in PMS-treated mice. Reductions in regional cerebral blood flow (CBF) after MCAO were improved by administration of PMS. Treatment with PMS increased phosphorylation of endothelial nitric oxide synthase (eNOS) in the ischemic core and penumbra region. In neuron-endothelial cell co-cultures, PMS promoted nitric oxide production after OGD. These findings indicate that SR inhibition acts as a neuroprotectant in the NVU and ameliorant of CBF abnormalities post-stroke. Thus, pharmacologic SR inhibition has potential clinical applications.

Antimicrobial Efficacy of Salvadora persica Extracts on a Monospecies Biofilm on Orthodontic Brackets In Vitro.

PURPOSE: The oral cavity is a rich ecosystem with a plethora of microorganisms, and different components of fixed orthodontic appliances may contribute to a shift in the balance of oral ecology. The purpose of this study was to investigate the antimicrobial potential of hexane and ethanol extracts of Salvadora persica on a monospecies biofilm model established on orthodontic brackets in vitro. MATERIALS AND METHODS: Streptococcus mutans biofilm was formed on mini diamond orthodontic brackets following three days of anaerobic incubation at 37˚C. The bacterial cell viability of this biofilm was measured after their exposure to saline, hexane extract of S. persica, ethanol extract of S. persica and 0.2% chlorhexidine using 3-(4, 5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay. On half of the brackets, the colony forming units (CFU) were counted. Both experiments were performed in triplicate. RESULTS: The absorbance values obtained from the MTS reduction assay after exposure to the different test agents showed a decline in the bacterial cell viability of the S. mutans biofilm as follows: chlorhexidine (+)0.05). The CFU counts of S. mutans obtained from chlorhexidine exposure were lower than from hexane and ethanol extracts. CONCLUSION: S. persica extracts were found to have antimicrobial effects on S. mutans biofilm established in vitro on orthodontic brackets suggestive of its potential use as an oral antimicrobial agent for orthodontic patients.

Catalytic photoinduced electron transport across a lipid bilayer mediated by a membrane-soluble electron relay.

Unidirectional photocatalytic electron transfer from a hydrophilic electron donor encapsulated in the interior of a liposome, to a hydrophilic electron acceptor on the other side of the membrane, has been achieved using the simple membrane-soluble electron relay 1-methoxy-N-methylphenazinium (MMP(+)). The total amount of photoproduct (>140 nmol) exceeds the number of moles of MMP(+) present (125 nmol), thus showing that the transport of electrons is catalytic.

Magnetic Resonance Imaging of Atherosclerosis Using CD81-Targeted Microparticles of Iron Oxide in Mice.

The goal of this study is to investigate the feasibility of using CD81- (Cluster of Differentiation 81 protein-) targeted microparticles of iron oxide (CD81-MPIO) for magnetic resonance imaging (MRI) of the murine atherosclerosis. CD81-MPIO and IgG- (Immunoglobulin G-) MPIO were prepared by covalently conjugating, respectively, with anti-CD81 monoclonal and IgG antibodies to the surface of the tosyl activated MPIO. The relevant binding capability of the MPIO was examined by incubating them with murine bEnd.3 cells stimulated with phenazine methosulfate (PMS) and its effect in shortening T2 relaxation time was also examined. MRI in apolipoprotein E-deficient mice was studied in vivo. Our results show that CD81-MPIO, but not IgG-MPIO, can bind to the PMS-stimulated bEnd.3 cells. The T2 relaxation time was significantly shortened for stimulated bEnd.3 cells when compared with IgG-MPIO. In vivo MRI in apolipoprotein E-deficient mice showed highly conspicuous areas of low signal after CD81-MPIO injection. Quantitative analysis of the area of CD81-MPIO contrast effects showed 8.96- and 6.98-fold increase in comparison with IgG-MPIO or plain MPIO, respectively (P < 0.01). Histological assay confirmed the expression of CD81 and CD81-MPIO binding onto atherosclerotic lesions. In conclusion, CD81-MPIO allows molecular assessment of murine atherosclerotic lesions by magnetic resonance imaging.

Effect of α-asarone on angiogenesis and matrix metalloproteinase.

α-Asarone is a main component of Acorus gramineus widely known as an oriental traditional medicinal stuff. A. gramineus has been known to have a variety of medicinal efficacies such as anti-gastric ulcer and anti-allergic activities, inhibition of histamine release and antioxidant effect. However, its effect on angiogenesis remains unclear. The aim of this study was to investigate the effect of α-asarone on induction of angiogenesis through modulation of matrix metalloproteinase (MMP). First of all, MTT assay was performed to evaluate the effect of α-asarone on cell viability using MTT assay, and then tube formation assay with human umbilical vein endothelial cells (HUVEC) in vitro and rat aorta ring assay ex vivo were carried out to elucidate its effect on angiogenesis. Treatment with α-asarone below 6µM showed no cytotoxicity in human fibrosarcoma cells (HT1080) and HUVEC. It was observed that α-asarone not only promotes tube formation of HUVEC but also induces angiogenesis of rat aorta. In addition, the effects of α-asarone on the expressions of protein and gene were evaluated using western blot analysis and RT-PCR assay. α-Asarone increased the expression levels of MMP-2 and MMP-9 stimulated by phenazine methosulfate (PMS) and phorbol 12-myristate 13-acetate (PMA) in HT1080. Especially, the expression level of antioxidant enzyme such as glutathione reductase was increased in the presence of α-asarone. Therefore, above findings suggest that α-asarone may play an important role in pathological diseases related to MMP and angiogenesis.