Determination of clethodim and its oxidation metabolites in crops by liquid chromatography with confirmation by LC/MS.

A method was developed for determination of the herbicide clethodim (C0) and its oxidation metabolites clethodim sulfoxide (C1) and clethodim sulfone (C2) in agricultural products. Upon extraction, both C0 and C1 were oxidized to C2 by m-chloroperoxybenzoic acid, and C2 was determined by liquid chromatography (LC). The C2 peak was confirmed by liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI). Recoveries of C0 from radish, tomato, onion, sweet potato, kidney bean, carrot, cabbage, and lettuce ranged from 91 to 118% following fortification at 0.05-1.0 ppm. The detection limit of C2 in crops was 0.01 ppm (S/N > 3). The fortified samples of onion, sweet potato, kidney bean, and carrot were confirmed by LC/MS (ESI), and the peak of C2 was detected.

[Contents of eleven phthalates and di(2-ethylhexyl) adipate in retail packed lunches after prohibition of DEHP-containing PVC gloves for cooking purposes].

Ten samples of retail packed lunches purchased from convenience stores were determined for 11 phthalates and di(2-ethylhexyl) adipate (DEHA) in August 2000, 2 months after the prohibition of DEHP-containing PVC gloves in Japan. Each homogenized sample was extracted with acetonitrile, partitioned with n-hexane, and cleaned up using Florisil and PSA columns. Phthalates in the extract were determined by GC/MS (SIM). The limits of detection were 14.9 ng/g for di(2-ethylhexyl) phthalate (DEHP) and 18.6 ng/g for dibutyl phthalate (DBP). Levels of phthalates in packed lunch samples were 45 to 517 ng DEHP/g (198 ng/g, average), ND to 90 ng DEHA/g, and ND to 10.0 ng BBP/g. Diisononyl phthalate (DINP) was detected in one sample at 76 ng/g. Average DEHP level in ten samples was 4% of that in 1999. The contents of other phthalates were also reduced. DBP was not detected in any sample. Recovery of deuterated isomers added as surrogates was 27.9% for DNP-d4, and 40.6 to 101.5% for the other phthalates.

Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing.

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.

Di(2-ethylhexyl) phthalate contamination of retail packed lunches caused by PVC gloves used in the preparation of foods.

Plasticizer contamination of foods sold in retail packed lunches and set lunches in restaurants was determined by GC/MS. The phthalate esters were as follows: diethyl, dipropyl, dibutyl, dipentyl, dihexyl, butylbenzyl, dicyclohexyl, di(2-ethylhexyl), dioctyl, diisooctyl (mixture of isomers) and diisononyl (mixture). Di(2-ethylhexyl) adipate was also determined. Sixteen packed lunches and ten set lunches were analysed, and in all samples the concentration of di(2-ethylhexyl) phthalate (DEHP) was the highest, at 0.80-11.8 mg/kg in packed lunches and 0.012-0.30 mg/kg in set lunches. The DEHP content of five packed lunches exceeded 1.85 mg, which is the EU tolerable daily intake (TDI) for a person of 50 kg body weight. Foodstuffs that were components of the packed lunches were taken from the factory at each step of preparation and phthalates were determined. For example, chicken contained 0.08 mg/kg DEHP when uncooked, 13.1 mg/kg after frying and 16.9 mg/kg after packing. Disposable PVC gloves used in the preparation of foods were apparently the source of high DEHP concentrations. The gloves used during cooking or packaging were sprayed with 68% (w/w) ethanol to sterilize them. PVC gloves from the factory contained 22 or 41% by weight of DEHP. To confirm the link with the contamination problem, samples of boiled rice, croquette and boiled dry radish were handled in the laboratory with PVC gloves containing 30% (w/w) DEHP. DEHP migration levels of 0.05 mg/kg in rice or 0.33 mg/kg in croquette, and 11.1 mg/kg in radish were found. The alcohol sprayed onto the gloves increased the migration of DEHP to 2.03 mg/kg in rice, 2.45 mg, kg in croquette, and 18.4 mg/kg in radish.

Simultaneous determination of residues of emamectin and its metabolites, and milbemectin, ivermectin, and abamectin in crops by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method was developed for the determination of emamectin and its metabolites (8,9-Z-isomer, N-demethylated, N-formylated, and N-methylformylated emamectin) in various crops. The analytes were extracted with acetone, cleaned up on cartridge columns (C18 and NH2), derivatized with trifluoroacetic anhydride and 1-methylimidazole, and determined by LC with fluorescence detection. Because radish inhibited the formation of the fluorescent derivatives, an additional Bond Elut PRS cartridge was used in the cleanup of Japanese radish samples. During sample preparation, N-formylated emamectin partially degraded to emamectin B1b and emamectin B1a, and the 8,9-Z-isomer partially degraded to N-demethylated emamectin. Therefore, emamectin and its metabolites were determined as total emamectin, i.e., their sum was estimated as emamectin benzoate. Their recoveries from most crops were approximately 80-110% with the developed method. The detection limits for the analytes in vegetables were 0.1-0.3 parts per trillion (ppt). The results for these compounds were confirmed by LC/mass spectrometry (LC/MS; electrospray ionization mode). Because the fluorescent derivative of emamectin was undetectable by LC/MS, the results for the analyte were confirmed by using a sample solution without derivatization. Limits of detection by LC/MS were similar to the fluorescence detection limits, 0.1-0.3 ppt in vegetables. In addition to the emamectins, milbemectin, ivermectin, and abamectin were also determined by the developed method.

Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time.

In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.

[Clean-up procedure with ion-exchange mini column in the analysis of flusulfamide in agricultural products by HPLC].

A clean-up procedure with an ion-exchange column in the analysis of flusulfamide by HPLC was examined. Pesticide in the sample was extracted with methanol following liquid-liquid partition with ethyl acetate. The ethyl acetate fraction was cleaned up with silica gel column chromatography. The eluate from the silica gel column was further cleaned up with SAX + PSA mini column, then determined by HPLC. Carotenoids and interfering peaks were removed by washing the combined mini columns with 10 mL of 20% acetone-containing n-hexane and 5 mL of acetone, and flusulfamide was eluted with 35 mL of acetone.

Liquid chromatographic determination of emamectin, milbemectin, ivermectin and abamectin in crops and confirmation by liquid chromatography-mass spectrometry.

Emamectin, milbemectin, ivermectin and abamectin are similar macrocyclic lactone chemicals used as an acaricides or parasiticides. We developed a simultaneous analytical method for determining the residual amounts of these compounds and emamectin metabolites in crops. A sample extracted with acetone was cleaned up with Bond Elut C18 and NH2. The sample was then fluorescence-derivatized with trifluoroacetic anhydride and 1-methylimidazole in acetonitrile. The analyte was measured by HPLC with fluorescence detection using an octadecylsilyl column with 3 microm particle size and gradient elution. In most crops, their recoveries by the developed method were ca. 80-110%. The detection limits of the analytes in vegetables were 0.1-0.3 ppt. Using the developed method, we surveyed the residues of these compounds in 20 commercial crops in Osaka, Japan. The result of the surveillance was that emamectin benzoate of 0.2-6.7 ppb was detected in nine cases and milbemectin of 16.7-279.3 ppb was detected in four cases. The detected samples were confirmed by LC-electrospray ionization (ESI) MS. The limit of detection by LC-ESI-MS was similar to the fluorescence detection level of 0.1-0.3 ppt in vegetables except for milbemectin.

Soluble intercellular adhesion molecule-1 and natural killer cell activity in gastric cancer patients.

Intercellular adhesion molecule-1 (ICAM-1), a molecule bound to the cell surface, is a ligand for leukocyte function antigen-1 (LFA-1), and the ICAM-1/LFA-1 system mediates various cell-cell interactions involved in immunity. Soluble ICAM-1 (sICAM-1) is a circulating substance and binds with LFA-1 of leukocytes, thus, making leukocytes less available for binding with cell surface ICAM-1 on target cells. The serum level of soluble ICAM-1 (sICAM-1) was found to be significantly elevated (p<0.01) in patients with early and advanced gastric cancer compared with healthy controls. Natural killer activity (NK activity) was assessed by measuring the cytotoxicity of peripheral blood mononuclear cells (PBMCs) for K562 cells. There was no significant difference in NK activity between gastric cancer patients and healthy controls when heat-inactivated fetal calf serum was used in assays. However, addition of patient serum significantly decreased (p<0.05) NK activity when the serum was from patients with advanced gastric cancer compared with healthy volunteers. Addition of anti-ICAM-1 monoclonal antibody 0 to 5.0 microg/ml caused little change in NK activity in healthy controls, but its addition at 10 microg/ml remarkably decreased NK-activity in gastric cancer patients, probably through antibody binding with ICAM-1 on target cells. In other experiments, liver metastasis was induced in mice by inoculation of colon 26 murine colon cancer cells. In vitro pretreatment of colon 26 cells with the anti-ICAM-1 monoclonal antibody significantly increased the number of metastatic nodules. These results suggest that both sICAM-1 and anti-ICAM-1 monoclonal antibody act as immunosuppressive factors by inhibiting the ICAM-1/LFA-1 system.

Serum levels of circulating intercellular adhesion molecule 1 in hepatocellular carcinoma.

BACKGROUND/AIMS: This is a comparative study of the relationship between. MATERIAL AND METHODS: Serum levels of circulating intercellular adhesion molecule-1 (cICAM-1) were measured by ELISA assay in four patients with chronic hepatitis (CH), 16 with liver cirrhosis (LC), 38 with hepatocellular carcinoma (HCC), and in nine healthy controls. RESULTS: No significant difference in cICAM-1 levels was observed between LC and HCC. The cICAM-1 level in HCC did not correlate with tumor markers but correlated well with tumor size. cICAM-1 level in HCC Stage III + IV was significantly higher than that of Stage I, and was higher in HCC with liver metastasis as opposed to HCC without metastasis. Furthermore, the cICAM-1 level of HCC decreased significantly after hepatectomy. CONCLUSION: These findings showed a close relationship between cICAM-1 and the progress of intrahepatic metastasis of HCC, indicating a possibility for using cICAM-1 as a prognostic marker.

[Acute renal failure in a case of paroxysmal nocturnal hemoglobinuria--a review of literature in Japan].

A case of paroxysmal nocturnal hemoglobinuria (PNH) associated with acute renal failure (ARF) is described. A 57-year-old female, who had been diagnosed as having PNH in 1983 at Kochi Medical School, was admitted to our hospital in April 1989, because of ARF with dark urine after a common cold. Hemodialysis was performed 5 times for ARF, and after almost completely recovering from ARF, she was discharged. The renal biopsy showed the deposition of hemosiderin in the proximal tubular cells. We surveyed fourteen case reports of PNH associated with ARF in Japan including our case. Ten cases developed ARF after infection causing hemolytic attack. Twelve of 14 cases were treated by hemodialysis and 13 cases were reversible. The histopathology of their renal biopsies revealed the deposition of hemosiderin in the proximal tubular cells in six of seven cases and tubular necrosis in three cases. These data showed that hemolytic attack and dehydration related to infection facilitated the induction of ARF.