In-source formation of N-acetyl-p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC-ECNICI-MS analysis

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M-PFB](-). In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen (N-acetyl-p-aminophenol, NAPAP-d(0); paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d(4); MW 155) in anhydrous acetonitrile and their GC-ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M-PFB](-) at m/z 150 from NAPAP-d(0) and m/z 154 from NAPAP-d(4), we observed highly reproducibly almost equally intense anions at m/z 149 and m/z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl-p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC-ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/z 149 to m/z 150 and of m/z 153 to m/z 154 decreased with increasing ion-source temperature in the range 100-250°C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI](-)) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC-EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC-MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.     

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis

Ubiquitin/proteasome‐mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing β2 and β6 conditional mutant proteasome subunits in a cell type‐specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell‐specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of β2 and β6 conditional mutants, specifically in the nurse cell compartment, led to a notable up‐regulation of sporadic apoptosis potency during early and mid‐oogenesis ‘checkpoints’, thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of β2 and β6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type‐dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.     

A tool for the qualitative comparison of membrane-embedded and detergent-solubilized membrane protein structures in projection

The calculation of projection structures (PSs) from Protein Data Bank (PDB)-coordinate files of membrane proteins is not well-established. Reports on such attempts exist but are rare. In addition, the different procedures are barely described and thus difficult if not impossible to reproduce. Here we present a simple, fast and well-documented method for the calculation and visualization of PSs from PDB-coordinate files of membrane proteins: the projection structure visualization (PSV)-method. The PSV-method was successfully validated using the PS of aquaporin-1 (AQP1) from 2D crystals and cryo-transmission electron microscopy, and the PDB-coordinate file of AQP1 determined from 3D crystals and X-ray crystallography. Besides AQP1, which is a relatively rigid protein, we also studied a flexible membrane transport protein, i.e. the l-arginine/agmatine antiporter AdiC. Comparison of PSs calculated from the existing PDB-coordinate files of substrate-free and l-arginine-bound AdiC indicated that conformational changes are detected in projection. Importantly, structural differences were found between the PSV-method calculated PSs of the detergent-solubilized AdiC proteins and the PS from cryo-TEM of membrane-embedded AdiC. These differences are particularly exciting since they may reflect a different conformation of AdiC induced by the lateral pressure in the lipid bilayer.     

Structure determination of channel and transport proteins by high-resolution microscopy techniques

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.     

Lipid synthesized by micro‐algae grown in laboratory‐ and industrial‐scale bioreactors

Tetraselmis sp. and Nannochloropsis oculata, cultivated in industrial‐scale bioreactors, produced 2.33 and 2.44% w/w lipid (calculated as the sum of fatty acid methyl esters) in dry biomass, respectively. These lipids contained higher amounts of neutral lipids and glycolipids plus sphingolipids, than phospholipids. Lipids of Tetraselmis sp. were characterized by the presence of eicosapentaenoic acid (that was located mainly in phospholipids), and octadecatetraenoic acid (that was equally distributed among lipid fractions), while these fatty acids were completely absent in N. oculata lipids. Additionally, lipids produced by 16 newly isolated strains from Greek aquatic environments (cultivated in flask reactors) were studied. The highest percentage of lipids was found in Prorocentrum triestinum (3.69% w/w) while the lowest in Prymnesium parvum (0.47% w/w). Several strains produced lipids rich in eicosapentaenoic and docosahexaenoic acids. For instance, docosahexaenoic acid was found in high percentages in lipids of Amphidinium sp. S1, P. parvum, Prorocentrum minimum and P. triestinum, while lipids produced by Asterionella sp. (?) S2 contained eicosapentaenoic acid in high concentration. These lipids, containing ω‐3‐long‐chain polyunsaturated fatty acids, have important applications in the food and pharmaceutical industries and in aquaculture.     

Characterizing associations and SNP-environment interactions for GWAS-identified prostate cancer risk markers--results from BPC3

Genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. However, whether these associations can be consistently replicated, vary with disease aggressiveness (tumor stage and grade) and/or interact with non-genetic potential risk factors or other SNPs is unknown. We therefore genotyped 39 SNPs from regions identified by several prostate cancer GWAS in 10,501 prostate cancer cases and 10,831 controls from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). We replicated 36 out of 39 SNPs (P-values ranging from 0.01 to 10⁻²⁸). Two SNPs located near KLK3 associated with PSA levels showed differential association with Gleason grade (rs2735839, P = 0.0001 and rs266849, P = 0.0004; case-only test), where the alleles associated with decreasing PSA levels were inversely associated with low-grade (as defined by Gleason grade <8) tumors but positively associated with high-grade tumors. No other SNP showed differential associations according to disease stage or grade. We observed no effect modification by SNP for association with age at diagnosis, family history of prostate cancer, diabetes, BMI, height, smoking or alcohol intake. Moreover, we found no evidence of pair-wise SNP-SNP interactions. While these SNPs represent new independent risk factors for prostate cancer, we saw little evidence for effect modification by other SNPs or by the environmental factors examined.     

Detection of the TNFSF members BAFF, APRIL, TWEAK and their receptors in normal kidney and renal cell carcinomas

In advanced renal cell carcinoma (RCC), surgery combined with systemic chemotherapy and immunotherapy have had limited effectiveness. Therapeutic modalities targeting VEGF, PDGF, and c-kit using tyrosine kinase inhibitors and m-TOR using specific biologic factors are in development. Therapeutic approaches targeting TNF-alpha have shown limited efficacy, while anti-TRAIL (TNFSF10) antibodies have shown enhanced activity. The presence and potential significance of other members of the TNFSF has not been investigated. Here, we assayed the TNFSF members APRIL, BAFF, TWEAK and their receptors (BCMA, TACI, BAFFR, Fn14) in 86 conventional type clear cell RCC, using immunohistochemistry and correlated our findings with histological data and, in a limited series, follow-up of patients. We observed a differential expression of these TNFSF ligands and receptors in cancerous and non-cancerous structures. BAFF was found in all RCC; APRIL expression is associated with an aggressive phenotype, correlating negatively with patients' disease-free survival, while TWEAK and its receptor Fn14 are heterogeneously expressed, correlating negatively with the grade and survival of RCC patients. This is the first study, presenting together the TNFSF members APRIL, BAFF, TWEAK and their receptors in different areas of normal renal tissue and RCC, suggesting a potential role of these TNFSF members in renal tumor biology.     

Characterization of an oxidoreductase from the arylamine N-acetyltransferase operon in Mycobacterium smegmatis

Mycobacterium?tuberculosis, the most successful bacterial pathogen, causes tuberculosis, a disease that still causes more than 2 million deaths per year. Arylamine N-acetyltransferase is an enzyme that is conserved in most Mycobacterium spp. The nat gene belongs to an operon that is important for the intracellular survival of M. tuberculosis within macrophages. The nat operon in Mycobacterium smegmatis and other fast-growing mycobacterial species has a unique organization containing genes with uncharacterized function. Here, we describe the biochemical, biophysical and structural characterization of the MSMEG_0308 gene product (MS0308) of the M. smegmatis nat operon. While characterizing the function of MS0308, we validated the oxidoreductase property; however, we found that the enzyme was not utilizing dihydrofolate as its substrate, hence we first report that MS0308 is not a dihydrofolate reductase, as annotated in the genome. The structure of this oxidoreductase was solved at 2.0 ? in complex with the cofactor NADPH and has revealed the hydrophobic pocket where the endogenous substrate binds.