Antitumor effect of memantine is related to the formation of the splicing isoform of GLG1, a decoy FGF­binding protein.

Drug repositioning is a strategy for repurposing the approved or investigational drugs that are outside the scope of the original medical indication. Memantine is used as a non­competitive N­methyl­D­aspartate receptor antagonist to prevent glutamate­mediated excitotoxicity in Alzheimer's disease, and is one of the promising agents which is utilized for the purpose of cancer therapy. However, the association between memantine and Golgi glycoprotein 1 (GLG1), an intracellular fibroblast growth factor receptor, in cancers has not yet been clarified. The present study analyzed the expression and location of GLG1 in tumor cells treated with memantine. Memantine was found to suppress the growth of malignant glioma and breast cancer cells in a concentration­dependent manner. The mRNA expression of GLG1 was upregulated in a concentration­dependent manner, and the splicing variant profiles were altered in all cell lines examined. The results of western blot analysis revealed an increase in the full­length and truncated forms of GLG1. Moreover, GLG1 spread in the cytosol of memantine­treated cells, whereas it localized in the Golgi apparatus in control cells. Since GLG1 functions as a decoy FGF receptor, the modulation of GLG1 may prove to be one of the mechanisms underlying the cancer­suppressive effects of memantine.

[Simultaneous Analysis of 7 Antiviral Agents in Chicken Tissues and Processed Products by LC-MS/MS].

Since amantadine, rimantadine, arbidol, laninamvir, oseltamivir, peramivir, and zanamivir may be used as antiviral agents to treat avian influenza, we herein developed a simultaneous assay using LC-MS/MS. This method was applied to chicken products (including yakitori (grilled chicken), fried chicken, chicken steak, and boiled eggs) as well as chicken tissues (muscle, fat, the liver, gizzards, and heart) and eggs.Samples were extracted with methanol-water (9 : 1), purified by a tandem column with an InertSep® MAX cartridge (upper part) and InertSep® MCX cartridge (lower part), and then measured by LC-MS/MS. The sample matrix had no effect on the identification of compounds. Chromatographic separation was performed on a ZIC-HILIC column using a mobile phase of 1% acetic acid solution and 1% acetic acid solution in acetonitrile, resulting in complete separation and other obstructive peaks from the sample matrices. An external solvent calibration curve was used for quantification.The application of the method to 6 samples of chicken tissues and eggs achieved good results of between 77.9 and 97.5% for trueness and between 1.7 and 9.2% for concurrent accuracy. The method was also applied to 9 samples of processed products, including grilled chicken and fried chicken, and achieved good results with true percentages ranging between 72.6 and 99.2% and concurrent accuracies between 3.0 and 11.2%. Therefore, the developed method may also be applied to processed products.The limit of quantification (LOQ) of the developed method was 0.01 mg/kg.The method was then applied to 42 types of commercial processed products, including yakitori, fried chicken, steamed chicken, chicken steak, and boiled eggs, and no antiviral agents were detected.Collectively, the present results confirmed that the method developed herein is applicable to not only chicken tissues, but also their processed products.

Ultrasound Modulates Fluorescence Strength and ABCG2 mRNA Response to Aminolevulinic Acid in Glioma Cells.

BACKGROUND: 5-Aminolevulinic Acid (5-ALA) photodiagnosis (PD) is an effective method to detect residual tumors during glioma surgery. However, fluorescence strength differs in malignant gliomas, and false-negative fluorescence may result in tumor residue. We investigated the effect of ultrasound on the intracellular level of protoporphyrin IX (PpIX) and expression level of ATP-binding cassette transporter 2 (ABCG2), which is thought to act as a membrane efflux pump of PpIX from cytosol. METHODS: The malignant glioma cell lines SNB19, U87MG, and T98G were used for in vitro experiments. Cultured cells underwent ultrasound irradiation (1 MHz, 3 W/cm2, duty cycle 10%) after administration of 5-ALA, and morphological changes in tumor cells were observed. PpIX levels and ABCG2 expression were evaluated. RESULTS: The glioma tumor cells showed transient morphological changes and detachment from the culture dish; however, most cells survived and reverted to their original morphology within 6 hours. PpIX expression levels increased in glioma cells after ultrasound irradiation, and the increase was earlier and greater than that for 5-ALA alone. ABCG2 expressions increased after 5-ALA administration but were lower in ultrasound-irradiated glioma cells. CONCLUSIONS: Ultrasound irradiation of malignant gliomas contributes to stronger 5-ALA-induced fluorescence by elevating intracellular PpIX levels. Suppression of ABCG2 expression by ultrasound may contribute to PpIX accumulation in glioma cells.

[Determination of Zilpaterol in Livestock Products by LC-MS/MS].

An analytical method for the determination of zilpaterol in livestock products was developed. The sample was stirred with n-hexane and n-hexane saturated acetonitrile, and zilpaterol in the sample was extracted with acetonitrile. The extract was cleaned up on a ODS cartridge column (1 g) and SCX cartridge column (500 mg). The LC separation was carried out using an Inertsil ODS-4 column and linear gradient elution with 0.1%formic acid and acetonitrile containing 0.1% formic acid as mobile phase. Detection of MS was carried out positive ion electrospray ionization mode. Average recoveries (n=5) of zilpaterol from 6 kinds of livestock products fortified at the MRLs (0.01 mg/kg) were 87.0-99.4%, and the relative standard deviations were 2.4-6.3%. The limits of quantitation were 0.01 mg/kg.

Determination of Diniconazole in Agricultural Products, Livestock and Marine Products by LC-MS/MS.

An LC-MS/MS method for the determination of diniconazole in agricultural products, livestock and marine products was developed. Diniconazole in agricultural products was extracted with acetone. The extract was concentrated and partitioned with n-hexane and 10% sodium chloride solution. Agricultural products such as grains and beans were defatted using n-hexane-acetonitrile. Livestock and marine products were extracted with a mixture of acetone and n-hexane, and the organic layer was evaporated to dryness. The residue was defatted using n-hexane-acetonitrile. Cleanup was carried out using a Florisil cartridge column and a graphitized carbon cartridge column for these samples. The LC separation was carried out on an Inertsil ODS-3 column with a linear gradient of 0.1% formic acid and acetonitrile containing 0.1% formic acid. MS was carried out in the positive ion electrospray ionization mode. The calibration curve was linear between 0.00125 to 0.00750 mg/L. Average recoveries (n=5) of diniconazole from 16 kinds of agricultural products, livestock and marine products fortified at the MRLs (0.01 ppm) were 88.3-108%, and the relative standard deviations were 0.5-5.1%. The limits of quantitation were 0.01 mg/kg.

Hyperthermotherapy enhances antitumor effect of 5-aminolevulinic acid-mediated sonodynamic therapy with activation of caspase-dependent apoptotic pathway in human glioma.

Sonodynamic therapy (SDT) has shown great potential as an approach for cancer treatment, and hyperthermotherapy (HT) is also a promising cancer therapy. Here, we investigate whether HT could improve the efficacy of SDT and to make a preliminary exploration on potential mechanism. Xenograft tumor was established in nude mice model, and SNB19 and U87MG glioma cell lines were utilized for in vitro experiment. Alamar blue assay was performed to assess cell viability. Optical microscope was used to characterize the morphology changes of the glioma cells induced by SDT and HT treatments. Apoptotic rate, mitochondrial membrane potential (MMP), and intracellular production of reactive oxygen species (ROS) were examined by flow cytometer. The cell apoptosis of tumor tissues were detected by TUNEL assay. Furthermore, the expression of apoptosis-related proteins was detected with Western blot in vitro and immunohistochemistry in vivo. SDT plus HT group could significantly reduce the cell viability with circular-cell morphological change, compared with SDT group, and cell viability was decreased depending on raise of 5-ALA concentration, ultrasound exposure time, and temperature. The results also indicate that HT increased a conspicuous apoptosis, ROS production, and a remarkable loss in MMP induced by 5-ALA-SDT in vitro. Meanwhile, our data also demonstrated that the combined treatment could significantly induce apoptosis and delay tumor growth in vivo. Furthermore, in both in vitro and in vivo experiments, SDT plus HT group expressed significantly higher protein levels of Bax and cleaved caspase-3, 8, and 9 compared to SDT, HT, and control groups and significantly lower protein level of bcl-2 than the other three groups, while the expression of these proteins was unchanged between HT and control groups. HT may provide an important promotion on 5-ALA-SDT and further propose that SDT in combination with HT is a new potential application for the treatment of human glioma.

Pattern recognition analysis of proton nuclear magnetic resonance spectra of extracts of intestinal epithelial cells under oxidative stress.

BACKGROUND: Mesenteric ischemia-reperfusion induces gut mucosal damage. Intestinal mucosal wounds are repaired by epithelial restitution. Although many different molecular mechanisms have been shown to affect cell metabolism under oxidative conditions, these molecular mechanisms and metabolic phenotypes are not well understood. Nuclear magnetic resonance (NMR) spectroscopic data can be used to study metabolic phenotypes in biological systems. Pattern recognition with multivariate analysis is one chemometric technique. The purpose of this study was to visualize, using a chemometric technique to interpret NMR data, different degrees of oxidant injury in rat small intestine (IEC-6) cells exposed to H2O2. METHODS: Oxidant stress was induced by H2O2 in IEC-6 cells. Cell restitution and viability were assessed at different H2O2 concentrations and time points. Cells were harvested for pattern recognition analysis of (1)H-NMR data. RESULTS: Cell viability and restitution were significantly suppressed by H2O2 in a dose-dependent manner compared with control. Each class was clearly separated into clusters by partial least squares discriminant analysis, and class variance was greater than 90% from 2 factors. CONCLUSION: Pattern recognition of NMR spectral data using a chemometric technique clearly visualized the differences of oxidant injury in IEC-6 cells under oxidant stress.

The glutamate AMPA receptor antagonist, YM872, attenuates cortical tissue loss, regional cerebral edema, and neurological motor deficits after experimental brain injury in rats.

A massive increase in extracellular glutamate is thought to contribute to brain damage after traumatic brain injury. We examined the neuroprotective effect of the AMPA receptor antagonist YM872 in a rat head injury model using the fluid-percussion procedure. Male Sprague-Dawley rats were subjected to right lateral (parasagittal) fluid-percussion brain injury or sham injury. At 15 min postinjury, they received either YM872 (20 mg/kg/h, 20 mg/3 mL) or normal saline (vehicle) intravenously for 4 h. The administration of YM872 significantly improved the composite neuroscore at 1 and 2 weeks postinjury (p < 0.05), and markedly reduced the volume of tissue loss in the injured cortex (p < 0.05). It also significantly reduced cerebral edema in the ipsilateral parietal cortex at 48 h postinjury (p < 0.01). These results indicate that the posttraumatic administration of YM872 may be neuroprotective by ameliorating cortical tissue loss and regional cerebral edema, and suggest the importance of AMPA receptors in traumatic brain damage involving secondary injury processes.

A simple and reproducible testing method for dialkyl phthalate migration from polyvinyl chloride products into saliva simulant.

We describe a method of mechanical agitation to determine rates of dialkyl phthalate migration from polyvinyl chloride (PVC) products into saliva simulant. The method consists of rotary shaking of a sample with 30 mL of saliva simulant (pH 7.0) at 35 degrees C in a 50 mL glass tube at 300 rpm for 15 min, then measuring the amount of dialkyl phthalate in the saliva simulant by HPLC with a UV detector. The migration rates of diisononyl phthalate (DINP), di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) from PVC plates containing about 45% (w/w) plasticizer (molded in our laboratory) were identical. However, the migration rates from molded plates containing 13% (w/w) DBP were almost double those of DINP and DEHP at the same ratios. In addition, the amounts of DINP that migrated in vitro after rotary shaking for 15 min were equivalent to those in vivo determined in saliva from volunteers who chewed plates for 60 min. The migration rates of dialkyl phthalates from 11 commercially available toys ranged from 15.6 to 85.2 micrograms/cm2/h [relative standard deviation (RSD), 3 to 12%].

Multiple immunostaining methods to detect traumatic axonal injury in the rat fluid-percussion brain injury model.

Immunohistochemistry using beta-amyloid precursor protein (APP) N-terminus antibodies is routinely used to detect traumatic axonal injury (TAI). The temporal and regional distributions of APP C- and N-terminus immunoreactivity were investigated in rats with experimental brain injury and compared to distribution of neurofilament (NF) immunoreactivity. Male Sprague-Dawley rats underwent right lateral fluid-percussion (FP) brain injury or sham injury. Six FP injury rats and two control rats were transcardially fixed with 10% formalin at 1, 6, 24, and 48 hours, and 1 and 2 weeks after injury and serial 6 microm-thick paraffin sections were prepared. At 6 hours after injury, APP C-terminus immunostaining labeled small neurons and axonal deposits in the injured parasagittal cortex, striatum, thalami, and dorsal medulla, whereas APP N-terminus and NF immunostaining showed few axonal deposits in the parasagittal cortex. At 24-48 hours post-injury, marked axonal damage, including axonal swelling and bulbs, was observed in the injured cerebral hemisphere, cerebellar white matter, and medulla. NF immunostaining was most sensitive for axonal damage in the injured deep cortical layers, cerebellum, and medulla. At 1-2 weeks after injury, APP N-terminus immunostaining mainly showed dot-like axonal profiles in the injured thalamus. APP C-terminus immunoreactivity may serve as an early marker of TAI, and the C-terminal fragments of APP may be involved in the evolution of TAI because C-terminal fragments of APP are neurotoxic and pro-apoptotic. Multiple immunostaining methods may be required to fully recognize the extent of TAI.