A. M. Abd El-Aty

Characterization of a stem cell population in lung cancer A549 cells

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.

A survey of ectoparasite infestations in stray dogs of Gwang-ju City, Republic of Korea.

This study was designed to investigate the incidence of ectoparasite infestation among stray dogs in Gwang-ju City, Republic of Korea. A total of 103 stray dogs collected in the Animal Shelter of Gwang-ju City from November 2003 to August 2005 were investigated in this study. Ectoparasites of one or more genera were detected in 45.6% (47 / 103) of the dogs examined for dermatologic lesions and/or skin scrapings (from 3-5 affected areas). Otodectes cynotis was found to be the most frequent parasite (22.3%, 23 / 103), followed by Sarcoptes scabiei var canis (19.4%, 20 / 103), Ctenocephalides canis (6.8%, 7 / 103), Demodex canis (4.9%, 5 / 103), and Trichodectes canis (1.0%, 1 / 103). Monospecific infestation was found in 83.0% (39 / 47) of the affected dogs, whereas concurrent infestations with 2 or more ectoparasites per animal were found in 17.0% (8 / 47) of the affected dogs. Trichodectes canis is reported for the first time in the Republic of Korea. Dogs less than 1 yr old were more heavily infected than other age groups (66.7%), and small-sized dogs of less than 3 kg body weight were more heavily infected than larger dogs (41.7%).

Comparative gene expression of intestinal metabolizing enzymes.

The purpose of this study was to compare the expression profiles of drug‐metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S‐transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S‐transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug‐metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs. Copyright

Detection of three herbicide, and one metabolite, residues in brown rice and rice straw using various versions of the QuEChERS method and liquid chromatography-tandem mass spectrometry

A single-run analytical method was developed to analyze the three herbicides azimsulfuron, bensulfuron-methyl, and mesotrione and its metabolite (4-methylsulfonyl-2-nitrobenzoic acid (MNBA)) in brown rice and rice straw using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Samples extracted using various versions of Quick, Easy, Cheap, Effective, Rugged, and Safe QuEChERS (original unbuffered, acetate (AOAC), and citrate (EN) buffered) methods gave poor recoveries of all the tested analytes in both matrices. The extraction efficiency was improved when primary-secondary amine (PSA) sorbent was removed from the purification step, with the best recovery being achieved for EN-QuEChERS, which was subsequently used throughout the study. Overall, a determination coefficients (R(2))⩾0.995 was achieved at matrix-matched calibration curves at various concentration ranges. The recovery rates at three fortification levels (limit of quantification (LOQ), 1/2 maximum residue limit (1/2MRL), and MRL) ranged from 78 to 114.5, with relative standard deviations (RSDs)<18% for all the tested analytes in both matrices. The LOQs for all herbicides were lower than the MRL set by the Ministry of Food and Drug Safety (MFDS), Republic of Korea. Field trials with the recommended, or double the recommended dose, revealed that the herbicides can safely be applied to rice, as no residues were detected in the harvested samples at 110days.

Maresin 1 attenuates NAFLD by suppression of endoplasmic reticulum stress via AMPK-SERCA2b pathway

Maresin 1 (MAR1), which is derived from docosahexaenoic acid biosynthesized by macrophages, has been reported to improve insulin resistance. Recently, it has been documented that MAR1 could ameliorate inflammation and insulin resistance in obese mice. These findings led us to investigate the effects of MAR1 on hepatic lipid metabolism. We found that MAR1 could stimulate AMP-activated protein kinase (AMPK), thereby augmenting sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) expression. This stimulation suppressed lipid accumulation by attenuating the endoplasmic reticulum (ER) stress in hepatocytes under hyperlipidemic conditions. Attenuation was mitigated by knockdown of AMPK or thapsigargin, a SERCA2b inhibitor. We also demonstrated that MAR1 administration resulted in increased hepatic AMPK phosphorylation and Serca2b mRNA expression, whereas hepatic ER stress was reduced in high-fat diet (HFD)-fed mice. Moreover, MAR1 treatment suppressed hepatic lipid synthesis, thereby attenuating hepatic steatosis in HFD-fed mice. In conclusion, our results suggest that MAR1 ameliorates hepatic steatosis via AMPK/SERCA2b-mediated suppression of ER stress. Therefore, MAR1 may be an effective therapeutic strategy for treating non-alcoholic fatty liver disease (NAFLD) via regulation of ER stress-induced hepatic lipogenesis.

Simultaneous determination of sulfoxaflor and its metabolites, X11719474 and X11721061, in brown rice and rice straw after field application using LC-MS/MS

ABSTRACT The present study was carried out to develop an analytical method for simultaneously detecting and quantifying sulfoxaflor and its metabolites (X11721061, X11719474) in brown rice and rice straw using liquid chromatography–tandem mass spectrometry. The parent compound and its metabolites were extracted and purified using original ‘QuEChERS’ method with modification. The matrix-matched calibration curve of sulfoxaflor and its metabolites in both matrices achieved good linearity with determination coefficients (R2) ≥0.9944. The overall recoveries of sulfoxaflor at two fortification levels (rice: 0.2 and 1.0 mg/kg; rice straw: 0.4 and 2.0 mg/kg) ranged from 97.37% to 107.71% with relative standard deviations (RSDs) <5%. On the other hand, the recoveries of both metabolites (X11721061 and X11719474) at 0.1 and 0.5 mg/kg (rice) and 0.2 and 1.0 mg/kg (rice straw) were satisfactory with values ranging from 83.70 % to 112.60% with RSDs <8%. During storage at −20°C, the analyte and its metabolites were stable for up to 87 days. The limits of quantification of 0.02 mg/kg were lower than the maximum residue limit (0.2 mg/kg) set by the Korean Ministry of Food and Drug Safety for brown rice. The method was successfully applied to paddy field treated with different programme schedules and a preharvest interval of 7 days was proposed based upon the current study. In sum, the developed method is accurate and reproducible for ensuring the reliable determination of sulfoxaflor (and its metabolites) in harvested rice grain and straw samples from the field. The residual level of parent compound does not seem to pose any hazardous effect and treated rice could be safely used for consumption.

Protectin DX ameliorates hepatic steatosis by suppression of endoplasmic reticulum stress via AMPK-induced ORP150 expression

Docosahexaenoic acid (DHA) and its bioactive compounds may have suppressive effects on inflammation, endoplasmic reticulum (ER) stress, and insulin resistance. Protectin DX (PDX), a double lipoxygenase product from DHA has shown a suppressive effect on inflammation and insulin resistance. However, the effects of PDX on ER stress and hepatic steatosis have not been elucidated yet. Herein we report that PDX could stimulate the AMP-activated protein kinase (AMPK) phosphorylation, thereby upregulating oxygen-regulated protein 150 (ORP150) expression in a dose-dependent manner. Treatment of HepG2 cells with PDX attenuated the palmitate-induced triglyceride accumulation through regulation of the sterol regulatory element-binding protein 1 (SREBP1)–mediated pathway. To deal with the pharmacological significance in the protective effects of PDX on hepatic steatosis, we performed in vivo experiments. In a mouse model, the PDX administration would alleviate the high-fat diet–induced hepatic steatosis and trigger the hepatic AMPK phosphorylation and ORP150 expression. PDX improved palmitate-induced and HFD-induced impairment of hepatic lipid metabolism and steatosis through suppression of ER stress via an AMPK-ORP150–dependent pathway.

Simple extraction method requiring no cleanup procedure for the detection of minocycline residues in porcine muscle and milk using triple quadrupole liquid chromatography-tandem mass spectrometry

A versatile analytical method was developed for simple detection of minocycline residues in porcine muscle and milk using liquid chromatography-triple quadrupole tandem mass spectrometry (LC/MS/MS) with electrospray ionization. Samples were extracted with a mixture of acetonitrile and ethyl acetate (2:1, v/v), and then defatted using n-hexane. No cleanup procedure was deemed necessary. Minocycline was separated on a reversed-phase analytical column using a combination of 0.1xa0% formic acid in water (A) and 0.1xa0% formic acid in acetonitrile (B) as the mobile phase. Matrix-matched calibration showed good linearity over a concentration range of 10–60xa0μg/kg with a determination coefficient (R2) of 0.9727. Fortified porcine and milk samples having concentrations equivalent to and double the limit of quantification (LOQxa0=xa010xa0ng/g), respectively, yielded recovery ranges between 83.02 and 8.03xa0% and relative standard deviations <18xa0%. Samples collected from a large market located in Seoul, Korea, tested negative for minocycline residue. These results show that a combination of acetonitrile and ethyl acetate can effectively extract minocycline from porcine muscle and milk without solid-phase extraction, a step usually required for cleanup before analysis. The developed method is simple, sensitive, and can be extrapolated to other food animal products that are rich in protein and fats.