Early specific free radical-related cytotoxicity of gas phase cigarette smoke and its paradoxical temporary inhibition by tar: An electron paramagnetic resonance study with the spin trap DEPMPO

Electron paramagnetic resonance (EPR) spin trapping studies demonstrated aqueous tar particulate matter (TPM) and gas phase cigarette smoke (GPCS) to behave as different sources of free radicals in cigarette smoke (CS) but their cytotoxic implications have been only assessed in CS due to its relevance to the natural smoking process. Using a sensitive spin trapping detection with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), this study compared the respective roles of CS- and GPCS-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation of filtered and unfiltered, machine-smoked experimental and reference cigarettes yielding a wide range of TPM yields. In buffer bubbled with CS the DEPMPO/superoxide spin adduct was the major detected nitroxide. Use of appropriate control experiments with nitric oxide radical (NO*) or carbonyl sulfide, and a computer analysis of spin adduct diastereoisomery showed that the hydroxyl radical (HO*) adduct of DEPMPO seen in GPCS-bubbled was rather related to metal-catalyzed nucleophilic synthesis than to direct HO* trapping. Unexpectedly a protective effect of TPM on murine 3T3 fibroblasts was observed in early (<3h) free radical-, GPCS-induced cell death, and carbon filtering decreased free radical formation, toxicity and lipid peroxidation in three cell lines (including human epithelial lung cells) challenged with GPCS. These results highlight an acute, free radical-dependent, harmful mechanism specific to the GPCS phase, possibly involving NO* chemistry, whose physical or chemical control may be of great interest with the aim of reducing the toxicity of smoke.     

Host use of a hemiparasitic plant: no trade-offs in performance on different hosts

To examine putative specialization of a hemiparasitic plant to the most beneficial host species, we studied genetic variation in performance and trade-offs between performance on different host species in the generalist hemiparasite, Rhinanthus serotinus. We grew 25 maternal half-sib families of the parasite on Agrostis capillaris and Trifolium pratense and without a host in a greenhouse. Biomass and number of flowers of the parasite were the highest when grown on T. pratense. There were significant interactions between host species and R. serotinus seed-family indicating that the differences in performance on the two hosts and without a host varied among the families. However, we found no significant negative correlations between performance of R. serotinus on the host species or between performance on the two hosts and autotrophic performance. Thus, the genetic factors studied here are not likely to affect the evolution of specialization of R. serotinus to the most beneficial host.     

Lack of beta1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype

The enteric nervous system arises mainly from vagal and sacral neural crest cells that colonise the gut between 9.5 and 14 days of development in mice. Using the Cre-LoxP system, we removed beta1 integrins in the neural crest cells when they emerge from the neural tube. beta1-null enteric neural crest cells fail to colonise the gut completely, leading to an aganglionosis of the descending colon, which resembles the human Hirschsprung's disease. Moreover, beta1-null enteric neural crest cells form abnormal aggregates in the gut wall, leading to a severe alteration of the ganglia network organisation. Organotypic cultures of gut explants reveal that beta1-null enteric neural crest cells show impaired adhesion on extracellular matrix and enhanced intercellular adhesion properties. They display migration defects in collagen gels and gut tissue environments. We also provide evidence that beta1 integrins are required for the villi innervation in the small intestine. Our findings highlight the crucial roles played by beta1 integrins at various steps of enteric nervous system development.     

Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest-derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.     

The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides: the mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver

Nonstereospecific addition of free radicals to chiral nitrones yields cis/trans diastereoisomeric nitroxides often displaying different electron spin resonance (ESR) characteristics. Glutathione peroxidase-glutathione (GPx-GSH) reaction was applied to reduce the superoxide adducts (nitrone/*OOH) to the corresponding hydroxyl radical (HO*) adducts (nitrone/*OH) of two nitrones increasingly used in biological spin trapping, namely 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide, and of 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO), a sterically hindered DEPMPO analogue. The method offered improved conditions to record highly resolved ESR spectra and by accurate simulation of line asymmetry we obtained clear evidence for the existence of previously unrecognized isomer pairs of cis- and trans-[DEPMPO/*OH] and [DIPPMPO/*OH]. Additional nitrone/*OH generation methods were used, i.e. photolysis of hydrogen peroxide and the Fenton reaction. We developed a kinetic model involving first- and second-order decay and a secondary conversion of trans to cis isomer to fully account for the strongly configuration-dependent behavior of nitrone/*OH. In the reductive system and, to a lower extent, in the Fenton or photolytic systems cis-nitrone/*OH was the more stable diastereoisomer. In various biologically relevant milieu, we found that the cis:trans-nitrone/*OH ratio determined right after the spin adduct formation significantly differed upon the GPx-GSH vs (Fenton or photolytic) systems of formation. This new mechanistic ESR index consistently showed for all nitrones that nitrone/*OH signals detected in the postischemic effluents of ischemic isolated rat livers are the reduction products of primary nitrone/*OOH. Thus, ESR deconvolution of cis/trans diastereoisomers is of great interest in the study of HO* formation in biological systems.     

Proteomic Changes of Alveolar Lining Fluid in Illnesses Associated with Exposure to Inhaled Non-Infectious Microbial Particles

Background

Hyperresponsiveness to inhaled non-infectious microbial particles (NIMPs) has been associated with illnesses in the airways. Hypersensitivity pneumonitis (HP) is considered to be the prototype for these NIMPs-related diseases; however, there is no consensus on the definitions or diagnostic criteria for HP and the spectrum of related illnesses.

Methods and Findings

In order to identify the possible diagnostic markers for illnesses associated with NIMPs in alveolar lining fluid, we performed a proteomic analysis using a two-dimensional difference gel electrophoresis on bronchoalveolar lavage (BAL) fluid from patients with exposure to NIMPs in the context of damp building-related illness (DBRI) or conditions on the borderline to acute HP, designated here as agricultural type of microbial exposure (AME). Samples from patients with HP and sarcoidosis (SARC) were included for reference. Results were compared to results of healthy subjects (CTR). Western blot was used for validation of potential marker proteins from BAL fluid and plasma. Protein expression patterns suggest a close similarity between AME and HP, while DBRI was similar to CTR. However, in DBRI the levels of the inflammation associated molecules galectin-3 and alpha-1-antitrypsin were increased. A novel finding emerging from this study was the increases of semenogelin levels in BAL fluid from patients with AME, HP and SARC. Histone 4 levels were increased in AME, HP and SARC. Elevated plasma levels of histone 2B were detected in HP and SARC, suggesting it to be a potential blood indicator for inflammatory diseases of the lungs.

Conclusions

In this study, the proteomic changes in bronchoalveolar lavage of DBRI patients were distinct from other NIMP exposure associated lung diseases, while changes in AME overlapped those observed for HP patient samples. Some of the proteins identified in this study, semenogelin and histone 4, could function as diagnostic markers for differential diagnosis between DBRI and HP-like conditions.     

Comparative osteology of the penguin‐like mid‐Cenozoic Plotopteridae and the earliest true fossil penguins,with comments on the origins of wing‐propelled diving

We compared the osteology of the late Eocene to early Miocene penguin‐like Plotopteridae from the North Pacific Basin with that of Paleocene stem group representatives of the Sphenisciformes and identified previously unrecognized similarities and differences. New data on the osteology of plotopterids, like the shape of the caudal end of the mandible, support a position of plotopterids outside the Suloidea, the clade formed by Sulidae, Phalacrocoracidae, and Anhingidae. However, as assumed by previous authors, the diving adaptations of plotopterids and sphenisciforms are likely to have evolved independently, and the resemblances in different parts of the postcranial skeleton therefore constitute one of the more striking examples of parallelism among tetrapods. We note that close relatives of both plotopterids and penguins forage by plunge diving. Whereas underwater locomotion of diving birds with a swimming ancestor is usually driven by the feet, we hypothesize that plotopterids and other wing‐propelled divers are more likely to have had volant ancestors that initiated diving by shallow plunges into the sea.     

Fluorimetric screening assay for protein carbonyl evaluation in biological samples

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.