Quantitation of N'-Nitrosonornicotine (NNN) in Smokers' Urine by Liquid Chromatography-Tandem Mass Spectrometry

The tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) is carcinogenic to humans (IARC Group 1). Assessing the tobacco smoke-related exposure to NNN by suitable biomarkers is of interest for risk evaluation. Recently, NNN and NNN-N-glucuronide have been quantified in urine of smokers. However, it is unknown what percentage of the absorbed dose of NNN is excreted as total NNN (sum of free and conjugated NNN) in urine of smokers. We developed a sensitive method based on liquid chromatography with tandem mass spectrometry with deuterium-labeled internal standard for the determination of total NNN in human urine. The limit of quantitation of the method was 2 pg/mL with a calibration line linear up to 256 pg/mL. In a study with 16 smokers in which the respiratory retention of NNN was measured through controlled smoking, we found that on average about 1% of the pulmonary NNN dose was excreted in 24 h urine as total NNN.

“Who's Winning the Human Race?” Cold War as Pharmaceutical Political Strategy 1

Between 1959 and 1962, Senator Estes Kefauver led a congressional investigation into the pricing practices of U.S. drug firms. As part of its defense, the industry mobilized the rhetoric of cold war and promoted the industry as a critical national asset in the global war against communism. The industry argued that any effort to undermine corporate innovation by inviting, as Kefauver proposed, greater government involvement in drug development threatened the public's health and invited socialism—in the form of socialized medicine—into the domestic political economy. This strategy proved critical to the industry's efforts to build political support for itself, particularly among the medical profession, and undermine Kefauver's reform agenda.

Phase 3 Randomized Trial on Larynx Preservation Comparing Sequential vs Alternating Chemotherapy and Radiotherapy

Both induction chemotherapy followed by irradiation and concurrent chemotherapy and radiotherapy have been reported as valuable alternatives to total laryngectomy in patients with advanced larynx or hypopharynx cancer. We report results of the randomized phase 3 trial 24954 from the European Organization for Research and Treatment of Cancer.

The Logic of Acceptance: Grounding Institutions on Agents’ Attitudes

In the recent years, several formal approaches to the specification of normative multi-agent systems (MASs) and artificial institutions have been proposed. The aim of this article is to advance the state of the art in this area by proposing an approach in which a normative MAS is conceived to be autonomous, in the sense that it is able to create, maintain and eventually change its own institutions by itself, without the intervention of an external designer in this process. In our approach the existence and the dynamics of an institution (norms, rules, institutional facts, etc.) are determined by the (individual and collective) acceptances of its members, and its dynamics depends on the dynamics of these acceptances. In order to meet this objective, we propose the logic ℒ ( Acceptance Logic ) in which the acceptance of a proposition by the agents qua members of an institution is introduced. Such propositions are true w.r.t. an institutional context and correspond to facts that are instituted in an attitude-dependent way. The second part of the article is devoted to the logical characterization of some important notions in the theory of institutions. We provide a formalization of the concept of constitutive rule , expressed by a statement of the form ‘ X counts as Y in the context of institution x ’. Then, we formalize the concepts of obligation and permission (so called regulative rules ). In our approach, constitutive rules and regulative rules of a certain institution are attitude-dependent facts which are grounded on the acceptances of the members of the institution.

Signalling pathways involved in 1-nitropyrene (1-NP)-induced and 3-nitrofluoranthene (3-NF)-induced cell death in Hepa1c1c7 cells

We previously reported that 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) elicited apoptotic cell death as well as non-apoptotic programmed cell deaths (PCDs) with paraptotic and necroptotic characteristics, respectively. In the present study, we have further confirmed and extended these findings. Flow cytometric analyses of 1-NP-exposed/3NF-exposed Hepa1c1c7 cells revealed that caspase-3 was only activated in the subpopulation of cells corresponding to that with classic apoptotic morphology. Immunocytochemical analysis indicated that leucocyte elastase inhibitor-derived DNaseII (LEI/L-DNaseII), apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were more clearly translocated to the nucleus following 3-NF exposure than after 1-NP. These 3-NF-induced changes in AIF and EndoG translocation were reduced by necrostatin-1, an inhibitor of necroptotic cell death. Both compounds lead to accumulation of lipid droplets and induced DNA damage. Activation of checkpoint kinase (CHK) 1 and H2AX, but not ataxia telangiectasia mutated and CHK2, were observed. Furthermore, inhibition of p53 using pifithrin-alpha reduced the cell death induced by both compounds, suggesting a role of DNA damage/CHK1/p53 pathway in the death process. 1-NP-induced cell death was in addition characterized by increased oxidative damage and intracellular accumulation of Ca(2+). These findings further support the notion that 1-NP elicited apoptotic cell death and PCD with paraptotic characteristics, while 3-NF induced apoptosis and a PCD with necroptotic features.

An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity

The objective of the present study was to develop a cellular phenotype assay for nucleotide excision repair (NER), using benzo[a]pyrene diol epoxide (BPDE) as model mutagen. Since in vitro exposure to BPDE may lead to DNA strand breaks resulting from both direct interaction with DNA and incisions introduced by the repair enzymes, we aimed to discriminate between both types of breaks using the comet assay and quantified the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells (PBMCs) with BPDE in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC). The assay was performed with a low (0.5 microM) and a high (2.5 microM) BPDE concentration. The individual NER capacity was defined as the amount of DNA damage induced by BPDE in presence of APC, diminished with the damage induced by BPDE and APC alone. First, the assay was applied to a NER-deficient human fibroblast cell line (XPA-/-) to validate the methodology. Lower repair capacity and a higher amount of BPDE-induced DNA adducts were observed for the XPA-/- fibroblasts as compared to the wild-type fibroblasts. Repeated experiments on PBMCs from four donors showed low intra-individual, intra-experimental and inter-assay variation for both concentrations, indicating the reliability of the method. To assess the inter-individual variation, the assay was applied to PBMCs from 22 donors, comparing the repair capacity after exposure to 0.5 microM (N = 10) and 2.5 microM (N = 12) BPDE. The repair capacity showed a higher inter-individual variation as compared to the intra-individual variation. Moreover, this difference was more pronounced using the low concentration. All these results indicate the adequacy of the method using this low concentration. Further improvement, however, should be recommended by applying the study with low BPDE concentration in a larger population and taking into account the relevant genotypes for NER.

Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy

Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA.carAB and PepA.cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by approximately 235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the -35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers.

Interaction of the HIV-1 frameshift signal with the ribosome

Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5' direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 +/- 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome.

Structural basis for the specificity of thymidylate kinases from human pathogens: implications for nucleotide analogues activation

End-stage renal disease in patients with Fabry disease: natural history data from the Fabry Registry

Fabry disease, an X-linked lysosomal storage disorder caused by deficiency of alpha-galactosidase activity, is associated with progressive loss of kidney function. This study was undertaken to characterize Fabry disease among patients who reached end-stage renal disease.