ERK-1 MAP kinase prevents TNF-induced apoptosis through bad phosphorylation and inhibition of bax translocation in HeLa cells

Extracellular signal-regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl-2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK-1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase-deficient form of ERK-1 (K71R) were more sensitive to TNF and CHX. In the ERK-1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK-1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK-1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK-1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase-8 inhibitor IETD-FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c-Jun N-terminal kinases activator, increased TNF-killing. The ERK-1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK-1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death. © 2009 Wiley-Liss, Inc

Simultaneous determination of opiates, methadone, cocaine and metabolites, cannabinoids in hair using GC-MS

A procedure is presented for simultaneous analysis of morphine, codeine, 6-monoacethylmorphine, methadone, cocaine, benzoylecgonine, ecgonine methylesther, cocaethylene, tetrahydrocannabinol, cannabidiol, and cannabinol, in hair, of drug of abuse, addicts. The method involves, decontamination in sodium dodecyl sulfate 1%, distilled water and methanol, pulverization in a ball mill,addition of deuterated internals standards, heat extraction in methanol. The extracts were evaporated to dryness and analyzed with gas/chromatography/mass spectrometry(GC/MS) before and after BSTFA/TMCS 1% silylation. The extracts were analyzed using electron impact GC/MS operating in SIM mode

Validation of an extraction and GC-MS quantification method of cocaine, methadone,and morphine in post-mortem adipose tissue

Abstract Adipose tissue is a complex biological matrix that necessitates several pre-analytical preparation steps to separate drugs and metabolites from the lipophilic matrix. A novel, sensitive, and specific gas chromatographic-mass spectrometric (GC-MS) method for the determination of cocaine (metabolites), methadone, and morphine in postmortem adipose tissue was developed, optimized, and validated. The method involves the aqueous acid extraction of analytes, alkalinization of the extract, solid-phase extraction with chloroform, and derivatization with BSTFA before GC-MS analysis. Deuterated compounds were used as internal standards for determination and quantification of analytes. Limits of detection were 0.005 µg/g for cocaine and cocaethylene, 0.02 µg/g for benzoylecgonine, 0.01 µg/g for ecgoninemethylester, 0.005 µg/g for methadone, and 0.01 µg/g for morphine. Linearity ranged from 0.1 to 1.000 µg/g for all analytes. Intra- and interday accuracy ranged from 70.6 to 105%, and intra- and interday precisions were less than 8.2% and 8.6%, respectively, for all analytes. The method showed a good recover